Document Sample

                                   p53: TRAFFIC COP AT THE
                                   CROSSROADS OF DNA REPAIR
                                   AND RECOMBINATION
                                   Sagar Sengupta*‡ and Curtis C. Harris*
                                   Abstract | p53 mutants that lack DNA-binding activities, and therefore, transcriptional activities,
                                   are among the most common mutations in human cancer. Recently, a new role for p53 has
                                   come to light, as the tumour suppressor also functions in DNA repair and recombination.
                                   In cooperation with its function in transcription, the transcription-independent roles of p53
                                   contribute to the control and efficiency of DNA repair and recombination.

                                  The tumour suppressor p53 functions primarily as a          bind to the NUCLEAR MATRIX. This binding affinity
(MMR). A DNA-repair process       transcription factor and can mediate its different down-    increases following genotoxic stress10 and is the rate-
that removes mispaired            stream functions by activating or repressing a large        limiting factor in the repair of higher-order DNA
nucleotides and insertion or      number of target genes1–4. p53 is one of the most com-      structures11.
deletion loops.
                                  monly mutated genes in human cancer, which leads to             In eukaryotes, the five main DNA-repair processes
NUCLEAR MATRIX                    the generation of a mutant protein with an altered          are NUCLEOTIDE-EXCISION REPAIR (NER), BASE-EXCISION REPAIR
A network of nuclear proteins     amino-acid sequence, usually in the DNA-binding             (BER), MMR, NON-HOMOLOGOUS END-JOINING (NHEJ) and
that provides a structural        domain5,6. Cellular proteins, such as mouse double          HOMOLOGOUS RECOMBINATION (HR)
                                                                                                                                    . Although only the
framework for organizing          minute-2 (MDM2), and viral proteins, such as human          transactivation-independent function of p53 is
                                  papilloma virus-16 E6 (HPV-16 E6), can degrade              involved in HR regulation, the tumour suppressor
NUCLEOTIDE-EXCISION REPAIR        wild-type p53 and limit its activity. Loss of function      modulates almost all other DNA-repair processes by
A DNA-repair process in which a   of wild-type p53 also occurs by the overexpression of       both transactivation-dependent and -independent
small region of the DNA strand    mutant p53 due to its dominant-negative effect7. In         pathways (FIG. 1). Therefore, p53 might function as the
that surrounds the UV-induced
                                  addition to its role in transcriptional regulation, tran-   ‘molecular node’2 that lies at the intersection of
DNA damage is recognized,
removed and replaced.             scriptionally independent functions of wild-type p53        upstream signalling cascades and downstream DNA-
                                  have a less well-known role in mediating at least some      repair and -recombination pathways. In this review, we
*Laboratory of Human              of its downstream effects, including apoptosis, DNA         will detail the transactivation-independent roles of
Carcinogenesis, National
Cancer Institute, National
                                  repair and DNA recombination.                               p53, discuss the integration of its transactivation-
Institutes of Health,                 Apart from its role as a sequence-specific transcrip-   dependent and -independent functions in DNA repair
37 Convent Drive,                 tion factor, whereby p53 binds to highly conserved p53-     and recombination, and try to understand how the
Building 37, Room 3068,           binding sequences that are generally present in the         abrogation of these functions of p53 can lead to cancer
Bethesda, Maryland,               promoters of its target genes2, p53 also binds ‘non-        in humans (summarized in TABLE 1).
20892-4255, USA.
 National Institute of            sequence specifically’ to various DNA structures8.
Immunology, Aruna Asaf Ali        Analysis using various DNA-binding assays revealed          Nucleotide-excision repair and p53
Marg, New Delhi-110067,           that the affinity of p53 for mismatched and bulged          The most versatile form of DNA repair, NER, operates
India.                            DNA is equal to, or even greater than, that of the          on damaged bases and disrupted base pairings that are
Correspondence to C.H.
                                  human MISMATCH REPAIR (MMR) complex, MSH2–                  caused by ultraviolet light (UV) or oxidative damage,             MSH6, under the same binding conditions9. Perhaps           which leads to changes in the structure of the DNA
doi:10.1038/nrm1546               more pertinently, within the in vivo context, p53 can       duplex. There are two NER pathways, which have partly

44   | JANUARY 2005 | VOLUME 6                                                                             

                                              distinct substrate specificities. Whereas global genomic            CYCLOBUTANE PYRIMIDINE DIMERS (CPDs) and pyrimidine
                                              repair (GGR) scans the entire genome, transcription-                (6-4)pyrimidone photoproducts ((6-4) PHOTOPRODUCTS).
                                              coupled repair (TCR) recognizes lesions that are associ-            During UV damage, expression of the UV-damage
                                              ated with transcription12,13. NER recognizes a broad                DNA-binding protein (UV-DDB) — which is com-
                                              range of different DNA lesions, including UV-induced                posed of two subunits, p127DDB1 and p48DDB2 — is
                                                                                                                  induced. It has been postulated that p48DDB2 is required
                                                                                                                  to activate the latent binding activity in p127DDB1, but
              Ionizing radiation                                UV, replication errors, HU                        once p127DDB1 acquires its activity, p48DDB2 is
                                  Changes in chromatin structure
                                                                                                                      Xeroderma pigmentosum complementation group C
                                                                                  ATR    RAD17                    (XPC)–RAD23B is a GGR-specific complex that is
                              complex                                                ATRIP                        involved in identifying disrupted base pairings. Binding
                                          P             ATM
                  BRCA1                                                           RAD9       Lesion, adduct       to (6-4) photoproducts by XPC–RAD23B is direct.
                                                        ATM                      RAD9                             However, binding to CPDs by XPC–RAD23B might
            CHK2                                                                         RPA
     P                                                                            ATRIP                           require the prior binding of the UV-DDB complex. XPC
                                                                             ATR    RAD17                 P       is not required during TCR; however, during this
                                            P                                                      CHK1           process, the stalled RNA polymerase II must be dis-
                                H2AX                                                                          P
                      P                         P                                       P                         placed and the base damage must be specifically recog-
                  P       53BP1     MDC1            P                          BLM       P                        nized by at least two TCR-specific factors, Cockayne
                                                                      P                                           syndrome group B (CSB) and CSA.
                                SMC1                                       H2AX     53BP1
                          P                                          P
                                                                                                                      The subsequent stages in GGR and TCR might be
                                                                                                                  identical. Lesion recognition is followed by the binding
                                                                                                                  of several other proteins: XPA and replication protein A
                                                                                                                  (RPA; both of which enhance base recognition), and the
                                                    P                  P
                                                    P         p53      P
                                                                                                                  transcription factor (TF)IIH subcomplex of RNA poly-
        NER                                                                                             NER       merase II (which unwinds DNA by the XPB and XPD
  BER                                                                                                             helicases in the vicinity of DNA damage). The two NER-
                          Transactivation-                                    Transactivation-                    specific endonucleases, XPG and ERCC1/XPF, bind and
                          independent                                         dependent             ?     BER
                          processes                                           processes                           cleave the DNA 3′ and 5′ to the site of the damage.
  NHEJ                                                                                                            Repair synthesis and subsequent replication (which is
         HR                                                                                             MMR       mediated by DNA polymerases α or ε, proliferating cell
                                                                                                                  nuclear antigen (PCNA), RPA and replication factor C
              recognition                                                                                         (RFC)) is followed by re-ligation.
                                                                                                                      Patients with defective NER suffer from xeroderma
                              Apoptosis          Cell-cycle-checkpoint arrest Apoptosis          Senescence       pigmentosum (XP), a rare autosomal-recessive disorder
                                                                                                                  that is characterized by sun sensitivity and premature
                                                                                                                  malignant skin carcinomas and neoplasms. XP is geneti-
                                                        DNA damage                                                cally heterogeneous: there are eight complementation
Figure 1 | p53 functions as a ‘molecular node’ in the DNA-damage response. The DNA-
                                                                                                                  groups designated XP-A to XP-G and XP-variant (XP-V).
damage signal from DNA double-strand breaks (DSBs; left-hand pathway) or replication stress                       Whereas XP-A to XP-G correspond to genetic alter-
(right-hand pathway) is primarily recognized by ATM (ataxia telangiectasia mutated) or the                        ations in one of seven genes that are involved in NER,
ATR–ATRIP (ataxia-telangiectasia-and-RAD3-related–ATR-interacting-protein) complex,                               XP-V is caused by defects in the post-replication-repair
respectively. Changes in chromatin structure can lead to the autophosphorylation-mediated                         machinery, but NER is not impaired. Therefore, XP-V
activation of ATM, which is subsequently recruited to the DSB. The site of damage contains the                    patients have normal excision repair, but show defective
MRN complex (which comprises RAD50, MRE11 (meiotic-recombination-11) and NBS1
                                                                                                                  DNA replication after UV irradiation. This is because
(Nijmegen breakage syndrome-1)) and BRCA1 (breast-cancer-susceptibility protein-1). H2AX
(histone-2A family, member X), 53BP1 (p53-binding protein-1), MDC1 (mediator of DNA-damage                        the gene that encodes XP-V, DNA polymerase ε, allows
checkpoint protein-1) and SMC1 (structural maintenance of chromosomes-1) are phosphorylated                       DNA synthesis across UV-induced TT-dimers in an
by ATM, and are the key proteins that are involved in transducing a DSB-induced signal. The                       error-free manner under normal conditions. Genes for
ATR–ATRIP complex is recruited to the replication forks by the single-stranded (ss)DNA–RPA                        XP groups A, B, D, F and G are required for both TCR
(replication protein A) complex. The RAD9–RAD1–HUS1 complex (RAD9 in the figure) and its                          and GGR, whereas genes for groups C and E are
loading factor RAD17 facilitate recognition of the stalled fork. BLM (Bloom syndrome protein) and
                                                                                                                  required for GGR, but not TCR17.
H2AX are phosphorylated in an ATR/CHK1-dependent manner in response to replication stress.
CHK1 and CHK2, the respective targets of ATR and ATM, also phosphorylate the transducer
                                                                                                                      So, does p53 affect NER in vivo? The loss of p53 in
proteins. The transducer proteins transmit the signal to effector proteins such as p53, which is                  human cells results in the reduced repair of UV-induced
phosphorylated at different, yet specific, residues by ATM, ATR, CHK1 and CHK2. p53 is involved                   DNA damage18–20. Intact cells that are heterozygous for
in cell-cycle arrest, DNA repair, apoptosis or senescence. p53 has roles that do not involve its                  mutant p53 have normal repair of (6-4) photoproducts,
transactivation functions during DNA repair — nucleotide-excision repair (NER), base-excision                     but decreased initial rates of CPD removal, compared
repair (BER), mismatch repair (MMR), non-homologous end-joining (NHEJ) — and homologous                           with normal cells19. Therefore, it is possible that p53
recombination (HR). The repair processes (NER, BER and MMR) are also partially influenced by
the transactivation function of p53. The transactivation-independent role of p53 during apoptosis
                                                                                                                  affects NER in vivo by affecting the function (or func-
has been recently described. The continuum between the transactivation-independent and -                          tions) of the proteins that are involved in this process. For
dependent functions might be dependent on the level of DNA damage and cell type. HU,                              example, p53 regulates the transcription of p48DDB2 and
hydroxyurea; UV, ultraviolet light.                                                                               XPC 21,22. In turn, p48DDB2 directly regulates p53 protein

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY                                                                                                    VOLUME 6 | JANUARY 2005 | 4 5

 Table 1 | The effect of p53 on DNA-repair and -recombination pathways
 Pathway              Type of               Damaging                         Dependence        Dependence                Effect of p53          Effect of
                      damage                agents/cause                     on p53-           on p53-                   on process-            process-specific
                                                                             transactivation   transactivation-          specific protein(s)    protein on
                                                                             function          independent function      functions              p53 functions
 Nucleotide-          CPD;                UV; cisplatin;                     Yes               Yes                       Yes                    Yes
 excision             (6-4) photoproducts 4-nitroquinoline oxide;
 repair (NER)                             and other oxidative
 Base-excision Single-base DNA              Oxidizing, methylating,          Controversial     Yes                       Yes                    Yes
 repair (BER)  damage (short-               alkylating, hydroxylating
               patch BER);                  agents; ionizing
               single-strand break          radiation; spontaneous
               (long-patch BER)             depurinations (short-
                                            patch BER); X-rays
                                            (long-patch BER)
 Mismatch             Mispaired             Slippage of polymerase           Yes               Yes                       Yes                    Yes
 repair               nucleotides;          during replication of
 (MMR)                insertion/deletion    repetitive sequences or
                      loops                 recombination; mutations
                                            in MMR genes
 Non-                 Double-strand         Ionizing radiation;              ND                Yes                       Yes                    Controversial
 homologous           break                 chemical agents such as
 end-joining                                neocarzinostatin
 Homologous Double-strand                   Ionizing radiation;              No                Yes                       Yes                    Yes
 recombination break                        chemical agents such as
 (HR)                                       neocarzinostatin
 CPD, cyclobutane pyrimidine dimers; ND, not determined; UV, ultraviolet light.

                                     levels after UV irradiation, which implies that there are        relaxation is achieved by the p53-mediated acetylation
BASE-EXCISION REPAIR                 mutual regulatory interactions between these two pro-            of histone H3. As p53 is not known to possess intrinsic
The main DNA-repair pathway          teins23. The recruitment of both XPC and TFIIH to the            histone acetyltransferase (HAT) activity, it recruits p300
that is responsible for the repair   sites of CPDs and (6-4) photoproducts is facilitated by          (and probably other HATs) to NER sites to carry out the
of apurinic and apyrimidinic
(AP) sites in DNA. BER is
                                     wild-type p53, but not the mutant protein24. p53-                acetylation functions (FIG. 2Bb).
catalyzed in four consecutive        inducible p48DDB2 is the key downstream factor that is               Whereas the role of p53 in GGR is well accepted32,
steps by a DNA glycosylase,          responsible for the transport of XPC to the sites of DNA         p53 might also facilitate TCR20,33,34, as human cells with
which removes the damaged            damage in irradiated cells25,26. This probably explains          compromised p53 function have defective TCR20,33.
base; an AP endonuclease (APE),
                                     why p48DDB2 and XPC, but not p53, colocalize to the              However, the role of p53 in TCR is controversial, as it
which processes the abasic site; a
DNA polymerase, which inserts        sites of UV-induced DNA damage27 (FIG. 2A).                      was reported that mammalian cells that are homozy-
the new nucleotide(s); and DNA           Apart from the above-mentioned transactivation-              gous for p53 mutations are deficient in GGR, but have
ligase, which rejoins the DNA        dependent functions, can p53 affect NER in a transacti-          normal TCR35–37. This apparent discrepancy between
strand.                              vation-independent manner? Purified p53 does not                 different studies might be due to the use of different
                                     seem to stimulate NER in a reconstituted in vitro NER            UV sources for irradiation. It has been shown that
JOINING                              assay28. However, we and others have shown that p53              although the loss of functional p53 significantly
The main DNA-repair pathway          can affect NER by binding to the TFIIH helicase sub-             reduces the efficiency of TCR due to exposure to poly-
that is used throughout the cell     units, XPB and XPD, thereby modulating their helicase            chromatic UVB (290–324 nm), p53 is essential for
cycle to repair chromosomal
                                     activity20,29. Subsequently, it was observed that XPB and        GGR but dispensable for TCR when cells are exposed to
DSBs in somatic cells. NHEJ is
error-prone because it leads to      XPD helicases are also components of the p53-medi-               ‘germicidal’, monochromatic UVC (254 nm), which is
the joining of the breaks without    ated apoptotic pathway29,30 (FIG. 2Ba). Mechanistically,         virtually absent from terrestrial sunlight38. However, the
a template.                          mutant p53 is a less efficient inhibitor of both XPB-            mechanistic basis for the role of p53 in UV-dependent
                                     and XPD-mediated helicase and transcription activi-              regulation of CPD removal remains unknown. Another
                                     ties. So, overall it does seem that p53 affects NER in a         component of the TFIIH complex that is necessary only
A DNA-recombination pathway,         transactivation-independent manner.                              for TCR, CSB, interacts with p53 in vitro and in vivo 20,39. It
which includes the repair of             As a further indication of its transactivation-inde-         has been proposed that the activated p53 sequesters and
DSBs, and which uses a               pendent role, p53 might function as a CHROMATIN-                 inactivates CSB protein, thereby stalling transcription
homologous double-stranded-
                                     ACCESSIBILITY FACTOR in NER. In response to localized            complexes, locally blocking chromatin condensation and
DNA molecule as a template for
the repair of the broken DNA.        subnuclear UV irradiation, after detection and initial           causing metaphase-specific fragility of human genes39.
                                     recognition of the transcription-associated lesion (by
CYCLOBUTANE PYRIMIDINE               TCR), p53-dependent chromatin relaxation occurs,                 Base-excision repair and p53
DIMER                                which subsequently extends over the entire genome31. It          BER protects mammalian cells from damage that is
(CPD). The main UV-induced
lesion that functions as a
                                     is postulated that global chromatin relaxation, in turn,         inflicted by cellular metabolism (including damage
structural block for                 leads to lesion detection over the entire genome by the          that results from methylating and oxidizing agents)
transcription and replication.       GGR system. Interestingly, the UV-induced chromatin              and by spontaneous depurinations. A large number of

46   | JANUARY 2005 | VOLUME 6                                                                                       

A                                                                                                           glycosylases with narrow, yet partially overlapping, sub-
    Coactivators                                       Coactivators                                         strate specificity, endonucleases and DNA polymerases
        p53                                                 p53                                             are involved in this repair process. BER is mediated by at
                                p48DDB2                                                      XPC
                                                                                                            least two pathways: a ‘short-patch-repair’ pathway,
      p53RE                                                p53RE
                                                                                                            which involves the repair of a single nucleotide, and a
                                                                                                            ‘long-patch-repair’ pathway, which involves repairing
                    p48DDB2                                                XPC
                                                                                                            2–15 nucleotides12,14.
                                                                                                                A recent study with apurinic or apyrimidinic (AP)
                                      p48DDB2      XPC                                                      endonuclease-1 (APE1/REF1) indicates that the transac-
                                                                                                            tivation function of p53 might be affected by changes in
                                                                                                            BER enzymes. APE1/REF1 associates with p53, potenti-
                                      p48DDB2      XPC                                                      ates DNA binding by p53 and enhances the transacti-
                                                                                                            vation, growth arrest and apoptotic functions of p53
                                               CPD                                                          in vivo. The downregulation of APE1/REF1 causes a
                                                                                                            reduction in the ability of p53 to transactivate its cog-
          TFIIH                                                                                             nate promoters40.
                    XPB                                                                                         So does p53, in turn, affect BER? The first hint of an
                                   Modulates activities
              XPD                                                        Modulates NER
                                   of XPB and XPD (in vitro)                                                in vivo role for p53 in BER was observed using extracts
                                                                                                            from cells that expressed wild-type p53 and that had an
                                                                                                            augmented BER response41. The onset of p53 facilitation
                                                                        TFIIH                               of BER in cells is determined by the level of DNA dam-
b          UV                                                                    XPB                        age. Whereas low doses of γ-irradiation or the platinum
                                                                                                            compound cisplatin (which causes the formation of
                          Lesion                           p48DDB2       CSB           p48DDB2              intrastrand crosslinks between adjacent guanines) result
                                                                                                            in an immediate enhancement of BER activity, high
                                                                                                            doses of the same DNA-damaging agents instead lead to
                                                                                                            a reduction in BER and the induction of p53-dependent
                                                                                                            apoptosis42. BER activity is associated with two distinct
                                                                                                            phases of cell-cycle progression in unstressed cells: the
                                                                                                            G0–G1 and G2–M phases. Exposure to γ-irradiation
         Condensed chromatin                         Transcription-associated lesion detection              enhanced the G0–G1 BER-associated activity and atten-
                                                                                                            uated the G2–M BER-associated activity. More perti-
                                                     XPC          p53          p300                         nently, the alteration in BER activity after γ-irradiation is
                                                                                            Acetylated H3
                                                                                                            regulated by wild-type p53, but not its tumour-derived
                                                                                                            mutant forms43.
                                                       p300             p300          p300                      Recently, Rotter and co-workers have also shown
                                                        p53             p53           p53                   that the effect of p53 on 3-methyladenine (3-MeAde)
                                                               XPC              XPC          XPC
                                                                                                            DNA glycosylase, the first enzyme that was identified
                                                                                                            in the short-patch-repair BER pathway, is dependent
                                                                                                            on the type of stress. Wild-type p53 downregulates
                                                   Global chromatin relaxation and                          the transcription and activity of 3-MeAde DNA gly-
                                                   subsequent detection on relaxed chromatin
                                                                                                            cosylase after exposure to nitric oxide (NO), thereby
Figure 2 | Role of p53 in nucleotide-excision repair. A | An example of the transactivation-                preventing the creation of a mutator phenotype 44
dependent function of p53. In the presence of specific co-activators, p53 functions as a                    (FIG. 3a) . By contrast, γ-irradiation elevates p53-
transcription factor by binding to p53-response elements (p53RE) to upregulate the expression of
                                                                                                            dependent 3-MeAde-DNA-glycosylase activity 44. The
the xeroderma pigmentosum complementation group C (XPC) and p48DDB2 genes during
nucleotide-excision repair (NER). XPC and p48DDB2 recognize and bind specific lesions on the DNA.
                                                                                                            presence of wild-type p53 enhanced the removal of
Whereas p48DDB2 is one of the components of the ultraviolet light (UV)-induced, UV-damage DNA-              oxidized bases, such as 8-oxoG, during exposure to
binding protein (UV-DDB), XPC–RAD23B is a global genomic repair (GGR)-specific complex that is              reactive oxygen species45 (FIG. 3b).
involved in identifying disrupted base pairing. UV-induced cyclobutane pyrimidine dimers (CPDs) are             So, is p53 absolutely required for BER or does it only
an example of the type of damage to which p48DDB2 transports XPC. However, physical binding                 have a stimulatory role in the process? A recent report
between XPC and p48DDB2 is yet to be reported. B | Examples of the transactivation-independent              indicated that BER intermediates can induce p53-inde-
functions of p53. p53 can modulate NER by two independent, yet possibly interlinked, mechanisms.
a | XPB and XPD helicases are subunits of the transcription factor (TF)IIH and can unwind ~30 base
                                                                                                            pendent cytotoxic and genotoxic responses46, which
pairs of DNA near the damaged site. In vitro, p53 binds to XPB and XPD, modulates their enzymatic           implies that the tumour suppressor might not have an
functions, and thereby probably affects NER. b | p53 can also function as a chromatin-accessibility         enzymatic role in the process. However, BER is stimu-
factor in vivo. During transcription-coupled repair (TCR), the UV-damage-induced DNA lesion is              lated by recombinant wild-type p53 in an in vitro
recognized by TCR-specific factors, such as CSB (a subunit of TFIIH), along with p48DDB2 (a subunit         reconstitution system. The p53-dependent stimulation
of UV-DDB). After the initial recognition of the transcription-associated lesion, p53-dependent             of BER is correlated with its ability to interact directly
chromatin relaxation occurs and extends over the entire genome, which then allows lesion detection
                                                                                                            with APE1/REF1, DNA polymerase β and 8-oxogua-
by the NER system. During global chromatin relaxation, p53 recruits the histone acetylase p300 to
NER sites to acetylate histone H3, thereby relaxing the chromatin and enhancing the detection of            nine glycosylase (OGG1)45,47. OGG1 has glycosylase
the lesion in the entire genome. Therefore, the presence of wild-type p53 helps in the optimal              activity that removes bases in DNA such as 8-oxoG and
utilization of NER.                                                                                         generates an AP site. The AP site is recognized by the

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY                                                                                              VOLUME 6 | JANUARY 2005 | 4 7

 a                                                                                                              Selenomethionine (SeMet), which is the main
                                                 DNA                                                        source of selenium in our diet, provides yet another
 NO-induced DNA damage                        glycosylase                Mutator phenotype
                                                                                                            connection between p53 and APE1/REF1, as it regulates
                                                                                                            p53 by an APE1/REF1-dependent redox mechanism.
                                                                                                            Incubation with SeMet activates p53, and enhances its
                                                                                                            transactivation potential through changes in its confor-
                                                                                                            mation due to APE1/REF1-mediated reduction50. The
                                                 p53                  DNA repair by BER                     DNA-binding activity of p53 is enhanced in the pres-
                                                                                                            ence of wild-type APE1/REF1, but not its mutant form.
                                                                                                            The activated p53 becomes capable of enhancing the
 b                                                                                                          DNA-repair machinery without any growth-suppres-
                                                 p53                                                        sive effects. As a result, the cells can tolerate higher doses
                                                                                                            of UV irradiation when grown in the presence of SeMet
                                                                                                            (FIG. 3b). Therefore, p53 can contribute to genomic sta-
                                                                                                            bility by effectively inducing DNA repair, but not per-
 Direct interaction,   Interaction with     Interaction with    Interaction with   Increase of 3-MeAde
 enhanced activity     DNA polymerase β,    OGG1, enhanced      SeMet via          DNA glycosylase and      manent growth arrest or apoptosis.
 of APE1/REF1          enhanced stability   removal of 8-oxoG   APE1/REF1          OGG1 activities during
                       of polymerase        from DNA                               γ-irradiation and ROS
                                                                                                            Mismatch repair and p53
                                                                                                            MICROSATELLITE INSTABILITY, which is characterized by
                                                                                                            mispaired nucleotides and insertions or deletions, is
                                                 BER                                                        the consequence of slippage of the DNA polymerase
Figure 3 | Role of p53 in base-excision repair. a | An example of the transcription-dependent               during the synthesis of repetitive sequences in replica-
effects of p53. Exposure to nitric oxide (NO) induces p53 to cause transcriptional repression of            tion or recombination. Methylated bases of the type
the 3-methyladenine (3-MeAde) DNA-glycosylase gene (MPG), which functions in the ‘short-                    O6-methylguanine (O6MeG), paired with a C or a T, are
patch-repair’ base-excision repair (BER) pathway (indicated by small arrows). Downregulation of             also corrected by MMR due to the combined action of
MPG transcription might prevent the development of the mutator phenotype that might result                  evolutionarily conserved repair-specific proteins.
from the increased accumulation of point mutations after NO-induced DNA damage. However,
p53 can stimulate other components of the BER pathway, thereby providing a ‘delicate balancing
                                                                                                            Among human germline mutations, the MMR proteins
act’ on BER activation. b | Examples of transactivation-independent functions of p53. The                   MutL homologue-1 (MLH1) and MutS homologue-2
transactivation-independent functions of p53 can be exerted by different, yet complementary,                (MSH2) together account for approximately half of all
mechanisms. p53 stimulates BER by interacting with APE1/REF1, 8-oxoguanine (8-oxoG)                         the hereditary non-polyposis colorectal cancer (HNPCC)
glycosylase (OGG1) and DNA polymerase β. p53 stimulates the activity of APE1/REF1 and                       patients12. However, recent evidence has indicated that
OGG1. p53–DNA-polymerase-β interaction enhances the stability of the polymerase. In vivo, the               the MMR system is not only involved in HNPCC, but in
APE1/REF1-mediated interaction of p53 with selenomethionine (SeMet) activates p53 and the
                                                                                                            a much broader spectrum of human cancers51.
DNA-repair machinery without affecting cell growth. In the presence of γ-irradiation or reactive
oxygen species (ROS), p53 induces the activities of 3-MeAde DNA glycosylase or OGG1,                            MMR proteins and p53 have reciprocal effects on
respectively, which demonstrates the stress-dependent effect of p53 on BER enzymes.                         each other’s activity. The ataxia telangiectasia mutated
                                                                                                            (ATM)-mediated stabilization of the mismatch repair
                                                                                                            protein MLH1–postmeiotic-segregation-increased-2
                                                                                                            (PMS2) heterodimer augments p53 activation during
                                     multifunctional enzyme APE1/REF1, which has both                       DNA damage52. This effect might have functional signif-
                                     endonuclease (APE1) and redox (REF1) activities. In vitro,             icance as male p53 –/– Msh2 –/– double-knockout mice
                                     p53 significantly enhances the excision of 8-oxoG                      show synergism in tumourigenesis53. So how does p53
                                     nucleotides from the DNA by enhancing the sequential                   affect MMR? p53 functions as a sequence-dependent
(6-4) PHOTOPRODUCT                   activities of OGG1 and APE1/REF1 (REF. 45).                            transactivator by binding to several response elements
A type of DNA lesion that            Interestingly, the level of DNA polymerase β is                        in the promoter region of human MSH2 (REF. 54). In fact,
accounts for one quarter of all
                                     markedly diminished in p53-mutant or p53-null cells.                   p53 and the transcription factor Jun are reported to syn-
the DNA distortions and that are
produced by moderate doses of        This indicates the possibility that a DNA-polymerase-                  ergize in the regulation of human MSH2 in response to
UV irradiation.                      β–p53 complex might affect the stability of the poly-                  UV exposure. The human MSH2–MSH6 complex
                                     merase and that this stabilizing activity is absent in                 enhanced the in vitro binding of p53 to DNA substrates
CHROMATIN-ACCESSIBILITY              p53-deficient cells48. The integrity of the N-, core and               bearing bulged bases by 3–4-fold55, which indicates that
A factor that allows the detection
                                     C-terminal domains of the tumour suppressor are all                    MMR proteins and p53 might function synergistically
and subsequent removal of            required for its correct localization and the various                  in cells. MMR and p53 can cooperate to control the sen-
bulky DNA adducts by ‘opening’       interactions of p53 with the different components of                   sitivity to the cytotoxic effects of cisplatin and limit its
the chromatin.                       the BER machinery 47.                                                  mutagenic potential in colon cancer cells56. During
                                        The question is still open whether p53-dependent                    S phase of the cell cycle, p53 and MSH2 bind efficiently
A mutational change that occurs      transactivation is necessary for its role in BER. In vitro             to early recombination intermediates, they colocalize
in the DNA, in which the             generated, transactivation-deficient, mutant p53 was                   with each other and with the recombination proteins
number of repeats of                 shown to be more efficient in modulating BER activity                  RAD50 and RAD51, and coexist within the same
microsatellites (short, repeated     than wild-type p53 (REF. 49). However, the same                        nuclear DNA–protein complexes. These results indicate
sequences of DNA) is different
from the number of repeats that
                                     p53(22,23) mutant has also been proposed to lose the                   that both MSH2 and p53 are recruited to the sites of
were in the DNA when it was          ability to stimulate BER due to the loss of DNA-poly-                  recombination-associated repair and possibly modulate
inherited.                           merase-β interactions47 (FIG. 3b).                                     the process57.

48   | JANUARY 2005 | VOLUME 6                                                                                            

                                       The association between the lack of MSH2 or MLH1             results together, it was proposed that p53 inhibits error-
                                    expression in humans and the presence of p53 muta-              prone, but not error-free, NHEJ 75.
                                    tions has been observed in sarcoma and non-small-cell               Analysis of double-knockout mice for individual
                                    lung carcinoma58,59. Of clinical interest is the correlation    NHEJ factors and p53 provide a fascinating look at the
                                    between a p53 or MSH2 mutation and the diminished               consequences of the failure to properly repair DSBs.
                                    survival rate of hepatocellular carcinoma and head              Mice that are deficient in Ku, XRCC4 or LIG4 suffer
                                    and neck cancer patients60,61. Microsatellite instability       extensive apoptosis of developing neurons, with Xrcc4 –/–
                                    and p53 mutations are associated with the abnormal              and Lig4 –/– mice showing late embryonic lethality78–80,
                                    expression of the MSH2 gene in adult acute leukaemia62.         which is dramatically rescued by the homozygous dele-
                                    The loss of p53 has relatively little effect on MMR-profi-      tion of p53 (REFS 81,82). However, the rescued double-
                                    cient cells, but confers substantial hypersensitivity to cis-   knockout Xrcc –/– p53 –/– and Lig4 –/– p53 –/– mice, as well
                                    plastin on MMR-deficient cells56, which indicates that          as the Ku80 –/– p53 –/– mice, succumb to progenitor-B-cell
                                    p53 and MMR cooperate in response to DNA damage.                lymphoma within 6–16 weeks after birth. The early
                                    However, it has been reported that p53 and MMR                  death is due to the inability of the p53 deletion to rescue
                                    mutations occur together in glioblastoma, but not in            lymphocyte development in mice that lack NHEJ fac-
                                    colorectal cancers63, which indicates a differential effect     tors, which results in improper V(D)J REARRANGEMENTS and
                                    of p53 on the MMR system in diverse organs.                     subsequent lymphomas bearing clonal and recurrent
                                                                                                    chromosomal rearrangements such as gene amplifica-
                                    Non-homologous end-joining and p53                              tions and translocations81–83. Therefore, the loss of NHEJ
                                    NHEJ is the predominant DNA-repair process in G1                factors results in unrepaired DSBs and causes an apop-
                                    phase, but is also active in all other phases of the cell       totic response by the p53-dependent activation, leading
                                    cycle64. In mammalian cells, most DNA double-strand             to embryonic lethality. Recent studies on double-
                                    breaks (DSBs) are repaired by NHEJ12,15. NHEJ is an             knockout (Artemis –/– p53 –/–) mice indicate that this
                                    ‘error-prone’ process as nucleotides at the break can be        NHEJ factor — Artemis (a nuclease that is mutated in a
                                    added or lost, and incorrect ends might be joined.              subset of human severe immunodeficient patients) —
                                    However, NHEJ is still highly utilized to repair DSBs as,       and p53 cooperate to suppress oncogenic N-Myc ampli-
                                    without it, large numbers of genetic alterations might          fication84. So, Artemis –/– p53 –/– mice show amplification
                                    accumulate. Limited inaccurate repair is tolerated in           of N-Myc and succumb to progenitor-B-cell tumours.
                                    mammalian cells, given that a high percentage of the
                                    genome does not code for proteins.                              Homologous recombination and p53
                                        NHEJ is carried out by the combined action of               HR is a fundamental and accurate process that is
                                    different proteins, including DNA-PK (DNA-dependent             involved in maintaining the integrity of the genome and
                                    protein kinase), XRCC4 (X-ray repair complementing              is conserved in all organisms. During HR, the sequence
                                    defective repair in Chinese hamster cells-4) and DNA            that is lost in one double-stranded-DNA molecule is
                                    ligase IV (LIG4). DNA-PK consists of a catalytic subunit        replaced by the physical exchange of a segment from the
                                    (DNA-PKcs) and a regulatory subunit Ku (a het-                  second identical copy of the DNA12. After the detection
                                    erodimer of Ku70 and Ku80). DNA-PK phosphorylates               of the DSB, the DNA is resected in the 5′→3′ direction
                                    and activates p53, which leads to apoptosis by transacti-       and the resulting 3′ single-stranded tail is coated initially
                                    vating downstream target genes65,66. However, other             by RPA, which, in turn, stimulates the polymerization of
                                    studies with a different strain of DNA-PKcs mice67 and a        the pro-recombinogenic protein, RAD51, and its subse-
                                    DNA-PK-defective cell line68 indicated that the p53-            quent interaction with DNA (this phase is referred to as
                                    mediated apoptotic response is intact, even in the              the presynaptic phase; FIG. 4). RAD52 stimulates the
V(D)J REARRANGEMENTS                absence of DNA-PK. p53 and DNA-PK form a complex                polymerization of RAD51. The polymerized RAD51
The process of rearrangement        in response to nucleoside analogues such as gemc-               searches the homology with the help of RAD54, a mem-
and fusions of variable gene (V),
                                    itabine, and the complex interacts with gemcitabine-            ber of the SWI2/SNF2 (SWI refers to yeast mating-type
diversity gene (D) and joining
gene (J) segments that generate     containing DNA. The stalling of the DNA-PK–p53                  switching, and SNF is an abbreviation for sucrose non-
functional immunoglobin genes.      complex at the site of drug incorporation stabilized and        fermenting) family of helicases, which can bind to
                                    activated p53, and induced apoptosis69.                         RAD51 stoichiometrically, stabilize the protein–DNA
HETERODUPLEX                            Can p53 directly influence NHEJ, with or without            complex and thereby stimulate strand invasion85. Once
A DNA duplex formed by the
association between two
                                    the interaction with the specific repair proteins that con-     the homology is located, the duplex is captured and the
homologous strands, each of         trol the process? The answer might be complicated.              RAD51 filament invades the homologous duplex to
which was previously hybridized     Both in vitro and in vivo studies show that wild-type p53       form the HETERODUPLEX structure (synaptic phase). A
to different complementary          protein is capable of rejoining DNA with DSBs, which            DNA-dependent ATPase, RAD54, promotes this process
strands. If the homology is less
                                    hints at its putative direct transactivation-independent        by transiently denaturing the DNA base pairs. Then,
than 100%, the heteroduplex
will contain base mismatches        role in NHEJ70,71. It has also been hypothesized that           heteroduplex-DNA extension and branch migration
that will require repair.           wild-type p53 can facilitate precise ligation72.                occurs, which constitutes the postsynaptic phase of the
                                    Conversely, it has also been shown that DSB rejoining           reaction. The intact double-stranded copy is used as a
HOLLIDAY JUNCTION                   increases with the loss of wild-type p53 (REF. 73). Using       template for DNA synthesis by DNA polymerases. DNA
A cruciform DNA structure that
is generated during the synaptic
                                    reporter-gene reconstitution, integration or in vitro end-      ligases join the newly synthesized fragments, and the
phase of homologous                 joining assays, wild-type p53 was reported to inhibit           HOLLIDAY JUNCTIONS are resolved by specific endonucleases
recombination.                      NHEJ74–77. In an attempt to bring these contradictory           that are known as resolvases.

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY                                                                                      VOLUME 6 | JANUARY 2005 | 4 9

                                                                              DNA double-strand          p53 controls HR both in vitro and in vivo, and its
                                                                              break                  inactivation results in enhanced spontaneous and
                                                                                                     stress-induced HR86–93. p53-deficient mice show an
                                                                              5′→3′ resection and    increased frequency of HR at different stages of devel-
                                                                              coating with RPA       opment94. Furthermore, p53 with hotspot mutations at
                                                                                                     codons 281, 273, 248, 175 or 143 was found to be
                                                                                                     severely defective, with recombination frequencies that
                                                                                                     were elevated up to 26-fold75. This is probably because
                                                                              Strand invasion by
                                                                                                     the cancer-derived p53 mutants (such as 273H) might
                                                                              polymerized RAD51      not recognize the Holliday junctions95. In a cell line that
                                                                                                     co-expresses mutant p53, the deletion frequencies on
                                                                                                     assay plasmids were 5–20 times higher than in cells that
                                                                                                     expressed wild-type p53 alone90. The p53-dependent
                                                                                                     downregulation of HR is maximal between the
                                                                                                     sequences of short homologies75. Critical amino-acid
                                                                    Branch    RAD54-promoted,
                                                                                                     residues that are required for p53-mediated transacti-
                                                                    migration RAD51-mediated
                                                                              heteroduplex           vation have been identified96. Mutations in these
                                                                                                     residues lead to the generation of a transactivation-
                                                       Heteroduplex                                  deficient mutant, p53(22,23). We and others have
                                                                                                     shown that p53 regulates both spontaneous and radia-
                                                                                                     tion-induced HR independently of its activities as a
                                                                              Resolution with        sequence-specific transcription factor and its subse-
                                                                              extension by           quent involvement in G1–S checkpoint control89,93,97–99.
                                                                              DNA polymerase             The effect of p53 on HR can be either by itself or in
                                                                                                     association with HR-specific proteins, or a combination
                                                     Ligases                                         of both. This effect on HR can either be on the hetero-
                                                                                                     duplex structure (as discussed in this section) or on
                                                                                                     Holliday junctions (in conjunction with RECQ HELICASES,
                                                                              Product of
                                                                              homologous             as discussed in the next section). Wild-type p53 alone
                                                                              recombination          might check the fidelity of HR events by specific mis-
                                                                                                     match recognition in the heteroduplex intermediates86,
                                                                                                     and restrain DNA exchange between imperfectly
                                      RPA         RAD51        RAD52       p53      RAD54
                                                                                                     homologous sequences, and so suppress tumourigenic
                                      Resolvase        Polymerase                                    genome rearrangements. The binding specificity of
                                                                                                     wild-type p53 to heteroduplex joint recombination
                                  Figure 4 | Role of p53 in homologous recombination.                intermediates depends on residues within the central
                                  Homologous recombination (HR) is a multistep process that is       DNA-binding core domain and the C-terminal
                                  mediated by the sequential accumulation of proteins. After         tetramerization regions of p53. Tetramerized p53 stably
                                  detection of the DNA double-strand break (DSB) and resection
                                  of the DNA in the 5′→3′ direction, RAD51 binds to single-
                                                                                                     binds to the strand-transfer regions, enabling the pro-
                                  stranded (ss)DNA and displaces replication protein A (RPA),        tein to exonucleolytically correct heteroduplex interme-
                                  which leads to RAD51 polymerization (this phase is referred to     diates early after strand invasion95.
                                  as the presynaptic phase). Although RAD52 stimulates the               p53 interacts with several proteins that are involved
                                  polymerization of RAD51, wild-type p53 can control the             in the HR machinery, and one of the most studied is its
                                  process in vivo (the steps in the HR process that are controlled   interaction with and the control of RAD51. We have
                                  by p53 are shown) by its direct binding to RAD51. Once the
                                                                                                     shown that the RAD51-mediated, elevated levels of HR
                                  homology search is successful, the duplex is captured and the
                                  RAD51 filament invades it to form the heteroduplex structure       that are seen in a host-cell reactivation assay can be con-
                                  (synaptic phase). RAD54, a DNA-dependent ATPase, stabilizes        trolled by a transactivation-deficient p53 (REF. 97,100).
                                  the RAD51–ssDNA complex, thereby promoting this process.           In vitro mapping studies indicate that RAD51 can bind
                                  RAD54 can itself bind to p53 and scan the heteroduplex, a          to two regions of p53; one between amino acids 94 and
                                  process that is probably regulated by p53 alone, or preferably     160, and a second between amino acids 264 and 315.
                                  targeted by RAD51. Depending on the extent of the damage,
                                                                                                     The binding site of p53 on RAD51 between amino acids
                                  the mismatch is either corrected exonucleolytically by p53 or it
                                  can restrain the exchange of imperfectly homologous
                                                                                                     125 and 220 is highly conserved between bacteria and
                                  sequences. Heteroduplex DNA extension and branch                   humans and is necessary for polymerization101,102. In this
                                  migration normally occurs during the postsynaptic phase of         context, it is pertinent to note that p53 also physically
                                  HR, and wild-type p53 can inhibit the RAD51-promoted               interacts with RAD54 and binds through its extreme
                                  branch-migration process. The effect of p53 on heteroduplex        C-terminal domain100 — the same region that also
RECQ HELICASE                     or RAD51-mediated branch migration has been shown                  senses mispairings within HR intermediates89. Wild-type
A family of evolutionarily        in in vitro studies. DNA polymerases use the intact copy to
                                  re-synthesize the deleted DNA sequences, DNA ligases join
                                                                                                     p53 (but not the tumour-derived mutant forms) abro-
conserved helicases, mutations
of which can lead to hereditary   the newly synthesized fragments and the Holliday junctions         gates wild-type RAD51 polymerization. Mutant RAD51
cancer-predisposition             are resolved by specific endonucleases that are known as           with reduced binding to p53 elevated HR and did not
syndromes in humans.              resolvases.                                                        inhibit the polymerization, even in the presence of

50   | JANUARY 2005 | VOLUME 6                                                                                    

                                     co-expressed wild-type p53 (REF. 100). The p53 (273H)         binding to the promoter110. At the post-transcriptional
                                     mutant shows a reduced capacity to associate with             level, the C-terminal region of p53 interacts with
                                     RAD51–DNA complexes, even under conditions that               either the N-terminal region of BLM or the C-termi-
                                     support DNA binding102,103. These results indicate that       nal region of WRN in vitro111–114. In vivo, both BLM
                                     p53 controls HR via direct interaction with and inhibi-       and WRN physically interact with p53 — possibly as
                                     tion of RAD51 (FIG. 4).                                       part of a multiprotein complex that is involved in
                                         Apart from the controlling function of p53 on             replication, recombination and repair97,112–115.
                                     RAD51 during the presynaptic phase, the two proteins              A functional linkage between p53 and the RecQ heli-
                                     can also interact during the synaptic phase of HR. It has     cases was obtained when it was reported that the ectopic
                                     been proposed that p53 and RAD51 can interact during          co-expression of p53 with WRN in p53-deficient cells
                                     or shortly after strand transfer. In vitro, the p53–RAD51     increases the level of transcriptionally active p53 levels.
                                     complex can recognize the heteroduplex joints more            This corresponded with an enhancement in the level of
                                     efficiently than p53 alone, and RAD51 is involved in tar-     the p53 downstream effector p21 and p53-mediated
                                     geting p53 to these joints. RAD51 stimulates the inher-       apoptosis114,116. During IONIZING RADIATION, p53-depen-
                                     ent 3′→5′ exonuclease activity of p53, which p53 uses         dent apoptosis is attenuated in BS and WS cell lines
                                     for the nucleolytic destruction of heteroduplexes that        and coincides with the physical interaction between p53
                                     encompass base mispairings103. Apart from the synaptic        and BLM or WRN112,113.
                                     phase, p53–RAD51 interaction might also function dur-             p53 can also affect the functions that are mediated by
                                     ing the postsynaptic phase of HR. For example, it has         RecQ helicases. Wild-type p53, and not the mutant form,
                                     been reported recently that, in vitro, wild-type p53, but     modulates the exonuclease activity of wild-type WRN,
                                     not the p53 mutant proteins p53(248P) and p53(273P),          but not of a C-terminal-deletion mutant of WRN117. In
                                     inhibits branch migration promoted by RAD51 (REF. 104)        addition to full-length, wild-type p53, a ten-amino-acid
                                     (FIG. 4).                                                     fragment of p53 (amino acids 373–383) can also attenu-
                                         It should be noted that many of the direct effects of     ate the unwinding of Holliday junctions by BLM or
                                     RAD51 on p53 have been obtained from in vitro experi-         WRN in vitro, whereas p53 phosphorylation at Ser376
                                     ments. There is a degree of controversy over whether          and Ser378 completely abolishes this inhibition. Some
                                     p53 can indeed act on extrachromosomal model sys-             of the cancer-derived p53 mutants reduce or abolish the
                                     tems, and it has been suggested that the regulation of        ability of both WRN and BLM to bind to and unwind
                                     HR by p53 is restricted to the highly ordered chromoso-       Holliday junctions115. This latter observation might have
                                     mal chromatin structure105. Moreover, it has also been        biological significance, because latent p53 is phosphory-
                                     hypothesized that instead of inhibiting RAD51-medi-           lated at Ser376 and is rapidly dephosphorylated by
                                     ated HR, p53 can protect mammalian cells from replica-        cellular stress118. Because the helicase function and
An event that is similar to          tion-associated DNA DSBs, possibly by suppressing the         nuclear localization of BLM or WRN are required for
crossing over and that can occur     formation of recombinogenic lesions106. These issues          the restoration of stalled replication forks during repli-
between sister chromatids at         need to be resolved by future experiments.                    cation stress97,117, it is perhaps not surprising that p53-
mitosis or at meiosis.
                                                                                                   knockout mouse embryo fibroblasts show easily
                                     Effect of p53 on RecQ helicases                               detectable DSBs during hydroxyurea (HU) treatment106.
A cytogenetic aberration             RecQ helicases represent a subfamily of 3′→5′ DNA             In vivo, p53 inactivation in BS cells causes a significant
involving symmetrical and            helicases that are highly conserved in evolution and are      increase in sister-chromatid exchanges compared with
asymmetrical interchanges            required for the maintenance of genomic integrity.            BS cells that contain wild-type p53, thereby showing
between chromatids.
                                     Mutations in three of the RecQ-helicase genes in              that p53 and BLM cooperatively affect HR and sister-
CROSSING OVER                        humans, BLM, WRN and RECQ4, lead to cancer-predis-            chromatid exchanges97.
A reciprocal exchange of genetic     position disorders — Bloom syndrome (BS), Werner                  The effect of p53 and BLM on HR is a direct conse-
information.                         syndrome (WS) and Rothmund–Thomson syndrome                   quence of complex intra-nuclear transport of these fac-
                                     (RTS), respectively107. BS patient cells have enhanced        tors. BLM is an early sensor of HU-mediated replication
A class of enzymes that are          rates of chromosomal instability and HR, as character-        stress. BLM and p53 colocalize and physically associate
involved in the regulation of        ized by the elevated rates of SISTER-CHROMATID EXCHANGES,     with each other and also with the HR factor RAD51.
DNA supercoiling.                    insertions, deletions, telomere associations and              HU-mediated relocalization of BLM to RAD51 foci
                                     QUADRIRADIALS . In vitro, recombinant BLM protein can         is p53 independent. However, BLM is required for
Radiation, such as X-rays and
                                     modulate CROSSING-OVER events, along with TOPOISOMERASE III   efficient p53 accumulation to these sites and for the
γ-rays (high-energy photons),        (REF. 108). WS cells also show aberrant HR. The defect is     physical association of p53 with RAD51 (REF. 97). In
that causes atoms to release         in the resolution stage of this process, which indicates      non-stressed asynchronous cultures, BLM is found in
electrons and become ions.                                                                                                           119,120
                                     that WRN is involved in the resolution of recombina-          PML NUCLEAR BODIES (PML NBs)             , which are regarded
                                     tion structures109.                                           as the protein-storage depots of the nucleus. Cells that
(PML NB). One type of nuclear            The inter-regulation between p53 and the RecQ             lack functional p53 accumulate lower amounts of BLM
speckle that contains several        helicases has been shown at various levels. For exam-         in PML NBs, whereas isogenic cell lines that have func-
proteins, including the              ple, the transcription factor Sp1-mediated expression         tional p53 show normal BLM accumulation under simi-
promyelocytic leukaemia              of WRN is modulated by p53 at the transcription               lar conditions113. These results could indicate that,
protein (PML). It is thought to
be the site of recruitment of
                                     level. Mutation of Sp1-binding sites in the WRN pro-          whereas BLM might be necessary for the transport of
various proteins and might also      moter prevented repression by p53, which indicates            p53 to sites of stalled DNA replication forks, p53 medi-
have a role in gene transcription.   that the p53–Sp1 complex might prevent Sp1 from               ates nuclear trafficking of BLM back to the PML NBs.

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY                                                                                     VOLUME 6 | JANUARY 2005 | 5 1

                                         The HR process is influenced by a delicate balance         p53: the ‘molecular node’
                                     between the pro-recombinogenic factors (for example,           DNA-damage-induced cell-cycle checkpoints are
                                     RAD51 and RAD54) and the anti-recombinogenic fac-              signal-transduction cascades that link the detection of
                                     tors (such as p53 and BLM). The activity of the RAD51          DNA damage to different downstream responses.
                                     recombinase during HR needs to be finely controlled as         There are three such gatekeepers in the eukaryotic cell
                                     elevated levels of RAD51 are observed in various               cycle: the G1–S checkpoint (which inhibits the pro-
                                     tumour cell lines, which indicates that hyper-recombi-         gression from G1 to S phase in the presence of DNA
                                     nation might have a role in tumourigenesis121,122.             damage, thereby preventing the replication of dam-
                                     Therefore, there need to be checks and balances to             aged DNA), the intra-S-phase checkpoint (which is
                                     allow the inherently accurate HR to proceed at opti-           induced as a result of damage that occurred during S
                                     mum efficiency while preventing hyper-recombination.           phase and to counter the lesions that had escaped the
                                     Several complexes among the anti-recombinogenic                G1–S checkpoint) and the G2–M checkpoint (which
                                     proteins, p53–BLM, p53–RAD51, p53–RAD54 and                    prevents the progression of cells from G2 to M phase,
                                     BLM–RAD54, have been described, which could possi-             thereby preventing mitosis in the presence of damaged
                                     bly modulate and fine-tune the response and help to            DNA). Cells respond to the cell-cycle checkpoints
                                     keep HR in balance97,100,107. Recent evidence from             either by transient growth arrest and the activation of
                                     knockout mice reinforces the idea of a functional rela-        DNA-damage-repair pathways or by the initiation
                                     tionship between p53 and RecQ helicases. Wrn-deficient         of apoptosis. Although the stabilization of p53 is the
                                     mice do not age prematurely in early generations123.           most fundamental event in the G1–S checkpoint (see
                                     However, Wrn –/– p53 –/– double-knockout mice show             below), recent results indicate that the tumour sup-
                                     accelerated tumourigenesis and develop a broader spec-         pressor also has important roles in the intra-S-phase
                                     trum of tumours than p53 –/– mice124. Moreover, the            checkpoint.
                                     Wrn –/– p53 –/– mice show an accelerated mortality rela-           The G1–S checkpoint is primarily induced by DSBs
                                     tive to Wrn +/– p53 –/– mice, which indicates that there are   (FIG. 1). ATM represents the primary phosphoinositide-
                                     functional interactions between the p53 and the Wrn            3-kinase-like kinase (PIKK) responder to ionizing-
                                     genes and their gene products. Recently, it has been           radiation-induced DSBs130. ATM phosphorylates key
                                     reported that in late generations, mice that are doubly        proteins in several signalling pathways either directly
                                     null for Wrn and the telomerase RNA component Terc             or indirectly through its in vivo substrate, checkpoint
                                     have telomerase dysfunction, which elicits a classic WS        protein-2 (CHK2; REFS 130–132). Phosphorylated H2AX
                                     premature-ageing-syndrome phenotype125. Crosses                (γ-H2AX) marks the actual sites of DNA damage133.
                                     between the Wrn–/– Terc –/– and p53–/– mice might pro-         The phosphorylation of p53 can be mediated directly by
                                     vide more answers regarding the interdependence of             ATM or indirectly via CHK2. So, one of the most
                                     p53 and WRN during the ageing process.                         important events in the G1–S checkpoint is the stabi-
                                         Uncontrolled hyper-recombination leads to                  lization and activation of p53 (REFS 131,134). These results
                                     genomic instability, including chromosomal deletions,          might be specific for normal human and mouse cells as
                                     translocations and gene amplifications. From the pre-          in some tumour cells, CHK2 might lose its role as a sta-
                                     sent literature, it is not obvious whether p53 loss alone      bilizing effector of p53 (REF. 135).
                                     can lead to hyper-recombination or whether p53 muta-               The intra-S-phase checkpoint is triggered by repli-
                                     tions, in conjunction with the loss of other downstream        cation stress that is caused by intrinsic events or by
                                     functions, lead to an increase in HR. In support of the        extrinsic genotoxic insults (for example, UV or HU)
                                     sole role of p53, it has been shown in both genetic and        that prevent the progression of replication forks
                                     cellular-knockout systems that the abrogation of               (FIG. 1). This pathway is primarily controlled by the
                                     wild-type p53 results in an increase in the frequency          ATR–ATRIP           (ataxia-telangiectasia-and-RAD3
                                     of spontaneous mutations and various chromosomal               related–ATR-interacting-protein) complex136. ATR
                                     aberrations including deletions, amplifications and            phosphorylates and activates CHK1, γ-H2AX137 and
                                     multiple translocations126–128. These results support the      transducer proteins such as BLM138. We have recently
                                     hypothesis that wild-type p53 alone is sufficient to facil-    shown that 53BP1 was required for the efficient accu-
                                     itate recombinational DNA repair and so, the mainte-           mulation of both BLM and p53 at the sites of stalled
                                     nance of genomic integrity. As an alternative to the           replication. The accumulation of BLM and p53 was
                                     above postulation, it has been reported that targeted          independent of γ-H2AX. CHK1 phosphorylates BLM
                                     inactivation of wild-type p53 by HR does not result in         during replication stress and phosphorylated CHK1
                                     ANEUPLOIDY . Increased rates of numerical or structural        was required for the accurate focal colocalization of
                                     chromosomal instabilities were not observed in p53-            53BP1 with BLM, and the consequent stabilization
                                     deficient cells. Rates of sister-chromatid exchange and        of BLM 139. p53 can be activated by ATR and/or
                                     HR were also unaffected by the p53 status in unstressed        CHK1-mediated phosphorylation140,141 or indirectly
The ploidy of a cell refers to the   cells. So, it has been argued that p53 disruption alone is     via transducer proteins such as BLM, which trans-
number of chromosome sets            not sufficient to cause chromosomal instability.               ports p53 to the sites of stalled replication forks97. So,
that it contains. Aneuploid          However, in this study only one single parental cancer         the signal during replication stress is transmitted
karyotypes are chromosome
complements that are not a
                                     cell line was used, and it is possible that this cell line     through the ATR–CHK1–BLM–p53 pathway 97,138.
simple multiple of the               could have carried changes in HR proteins, which could         This results in p53 stabilization and, consequently,
haploid set.                         have masked the effects of changes in the p53 status.          the transcriptional attenuation of p53142.

52   | JANUARY 2005 | VOLUME 6                                                                                    

                                                So, p53 functions as a ‘molecular node’2 whereby the                               stabilization, which is dependent on post-transla-
                                             signals converge in response to either DSBs or stalled                                tional modification, and functions as a sequence-
                                             replication forks. p53 decides which effector role to                                 dependent transcription factor. It thereby activates a
                                             assume, probably depending on the cell-cycle condi-                                   set of genes that arrest the cell cycle, so that the DNA-
                                             tions and the type and extent of DNA damage. p53 can                                  repair processes can successfully correct the lesion.
                                             function either as a transactivator of genes that are                                 During this step, p53 can also interact and modulate
                                             involved in cell-cycle arrest, apoptosis or senescence,                               the different repair-process-specific proteins. If the
                                             or as a modulator of DNA-repair and -recombination                                    DNA damage persists or is irreparable, apoptotic-
                                             processes via either its transactivation-independent or                               specific genes are induced by p53, which leads to cell
                                             -dependent functions. During the transactivation-                                     death. So, p53 serves as the ‘guardian of the
                                             independent process, p53 interacts with proteins that                                 genome’143 by functioning as the ‘cellular rheostat’
                                             are involved in HR, NHEJ, MMR, BER and NER,                                           that modulates its multi-variant functions on the
                                             thereby adding another aspect to its known role as a                                  basis of the specific in vivo situation (FIG. 1).
                                             genome-surveillance protein (FIG. 1).                                                     Although recent efforts have clarified the transactiva-
                                                                                                                                   tion-independent functions of p53 during DNA
                                             p53: the cellular rheostat                                                            repair and recombination, many questions remain.
                                             The p53 response depends on its subcellular localiza-                                 Mechanistically, it would be helpful to know whether
                                             tion with respect to the site of DNA damage, the cell-                                and how p53 interacts with other, as-yet-unknown,
                                             cycle status at the time of DNA damage and the dose                                   components or auxiliary proteins that are involved in
                                             and duration of the genotoxic stress. It can be envis-                                the different repair processes. Because p53 has an effect
                                             aged that when the extent of DNA damage is low, the                                   on DNA repair, replication and recombination, the
                                             latent pool of p53, whether it is unmodified or post-                                 multiprotein complexes are present at these sites and are
                                             translationally modified, might interact with the                                     coordinated by mutual interactions and p53 activity.
                                             DNA-repair machinery — either alone or in combi-                                      The elucidation of the complexes in vivo, their hierarchy
                                             nation with other repair-specific factors. The advan-                                 of importance, and the kinetics and dynamics of associ-
                                             tage of such a graded response is that it prevents an                                 ation with the DNA lesion should all be the subject of
                                             ‘overreaction’ in response to the low level of DSBs                                   future research.
                                             and DNA distortions that might arise during the cell                                      Finally, the focus of this review has been to integrate
                                             cycle or in response to low-level exposure to media-                                  the transactivation-independent functions of the
                                             tors of DNA damage. During the response to DNA                                        tumour suppressor during DNA repair and recombi-
                                             damage, the upstream signalling process carries out a                                 nation. Recent research has shown that the transactiva-
                                             key role by recognizing the damage and post-transla-                                  tion-independent function of p53 is also involved
                                             tionally modifying p53. When the extent of DNA                                        during apoptosis144. It could be imagined that there is
                                             damage exceeds the level that can be successfully han-                                mutual regulation between the different transactiva-
                                             dled by p53 alone, the tumour suppressor undergoes                                    tion-independent functions of p53.

1.  Zhao, R. et al. Analysis of p53-regulated gene expression                higher-order DNA structure. Biochim. Biophys. Acta 1446,         22. Adimoolam, S. & Ford, J. M. p53 and DNA damage-
    patterns using oligonucleotide arrays. Genes Dev. 14,                    181–192 (1999).                                                      inducible expression of the xeroderma pigmentosum
    981–993 (2000).                                                      12. Hoeijmakers, J. H. Genome maintenance mechanisms for                 group C gene. Proc. Natl Acad. Sci. USA 99, 12985–12990
2. Vogelstein, B., Lane, D. & Levine, A. J. Surfing the p53                  preventing cancer. Nature 411, 366–374 (2001).                       (2002).
    network. Nature 408, 307–310 (2000).                                 13. Friedberg, E. C. How nucleotide excision repair protects             References 21 and 22 show how wild-type p53
3. Mirza, A. et al. Global transcriptional program of p53 target             against cancer. Nature Rev. Cancer 1, 22–33 (2001).                  facilitates NER by acting as a sequence-dependent
    genes during the process of apoptosis and cell cycle                 14. Dianov, G. L., Sleeth, K. M., Dianova, I. I. & Allinson, S. L.       transactivator of genes that encode DNA-repair
    progression. Oncogene 22, 3645–3654 (2003).                              Repair of abasic sites in DNA. Mutat. Res. 531, 157–163              proteins.
4. Polyak, K., Xia, Y., Zweier, J. L., Kinzler, K. W. & Vogelstein, B.       (2003).                                                          23. Itoh, T., O’Shea, C. & Linn, S. Impaired regulation of tumor
    A model for p53-induced apoptosis. Nature 389, 300–305               15. Lieber, M. R., Ma, Y., Pannicke, U. & Schwarz, K.                    suppressor p53 caused by mutations in the xeroderma
    (1997).                                                                  Mechanism and regulation of human non-homologous DNA                 pigmentosum DDB2 gene: mutual regulatory interactions
5. Levine, A. J., Momand, J. & Finlay, C. A. The p53 tumour                  end-joining. Nature Rev. Mol. Cell Biol. 4, 712–720 (2003).          between p48DDB2 and p53. Mol. Cell. Biol. 23, 7540–7553
    suppressor gene. Nature 351, 453–456 (1991).                         16. Sancar, A., Lindsey-Boltz, L. A., Unsal-Kaccmaz, K. & Linn, S.       (2003).
6. Hollstein, M., Sidransky, D., Vogelstein, B. & Harris, C. C.              Molecular mechanisms of mammalian DNA repair and the             24. Wang, Q. E. et al. Tumor suppressor p53 dependent
    p53 mutations in human cancers. Science 253, 49–53                       DNA damage checkpoints. Annu. Rev. Biochem. 73, 39–85                recruitment of nucleotide excision repair factors XPC and
    (1991).                                                                  (2004).                                                              TFIIH to DNA damage. DNA Repair (Amst.) 2, 483–499
7. Ko, L. J. & Prives, C. p53: puzzle and paradigm. Genes Dev.           17. Tang, J. & Chu, G. Xeroderma pigmentosum                             (2003).
    10, 1054–1072 (1996).                                                    complementation group E and UV-damaged DNA-binding               25. Fitch, M. E., Nakajima, S., Yasui, A. & Ford, J. M. In vivo
8. Liu, Y. & Kulesz-Martin, M. p53 protein at the hub of cellular            protein. DNA Repair (Amst.) 1, 601–616 (2002).                       recruitment of XPC to UV-induced cyclobutane pyrimidine
    DNA damage response pathways through sequence-                       18. Smith, M. L., Chen, I. T., Zhan, Q., O’Connor, P. M. &               dimers by the DDB2 gene product. J. Biol. Chem. 278,
    specific and non-sequence-specific DNA binding.                          Fornace, A. J. Jr. Involvement of the p53 tumor suppressor           46906–46910 (2003).
    Carcinogenesis 22, 851–860 (2001).                                       in repair of u.v.-type DNA damage. Oncogene 10,                  26. Wang, Q. E., Zhu, Q., Wani, G., Chen, J. & Wani, A. A.
9. Lee, S., Elenbaas, B., Levine, A. & Griffith, J. p53 and its              1053–1059 (1995).                                                    UV radiation-induced XPC translocation within chromatin is
    14 kDa C-terminal domain recognize primary DNA damage                19. Ford, J. M. & Hanawalt, P. C. Expression of wild-type p53 is         mediated by damaged-DNA binding protein, DDB2.
    in the form of insertion/deletion mismatches. Cell 81,                   required for efficient global genomic nucleotide excision            Carcinogenesis 25, 1033–1043 (2004).
    1013–1020 (1995).                                                        repair in UV-irradiated human fibroblasts. J. Biol. Chem.        27. Fitch, M. E., Cross, I. V. & Ford, J. M. p53 responsive
    A key study that shows the in vitro binding of                           272, 28073–28080 (1997).                                             nucleotide excision repair gene products p48 and XPC, but
    wild-type p53 to abnormal DNA structures.                            20. Wang, X. W. et al. p53 modulation of TFIIH-associated                not p53, localize to sites of UV-irradiation-induced DNA
10. Jiang, M. et al. p53 binds the nuclear matrix in normal cells:           nucleotide excision repair activity. Nature Genet. 10,               damage, in vivo. Carcinogenesis 24, 843–850 (2003).
    binding involves the proline-rich domain of p53 and                      188–195 (1995).                                                  28. Sancar, A. DNA repair in humans. Annu. Rev. Genet. 29,
    increases following genotoxic stress. Oncogene 20,                   21. Hwang, B. J., Ford, J. M., Hanawalt, P. C. & Chu, G.                 69–105 (1995).
    5449–5458 (2001).                                                        Expression of the p48 xeroderma pigmentosum gene is              29. Leveillard, T. et al. Functional interactions between p53 and
11. Aranda-Anzaldo, A., Orozco-Velasco, F., Garcia-Villa, E. &               p53-dependent and is involved in global genomic repair.              the TFIIH complex are affected by tumour-associated
    Gariglio, P. p53 is a rate-limiting factor in the repair of              Proc. Natl Acad. Sci. USA 96, 424–428 (1999).                        mutations. EMBO J. 15, 1615–1624 (1996).

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY                                                                                                                           VOLUME 6 | JANUARY 2005 | 5 3

30. Wang, X. W. et al. The XPB and XPD DNA helicases are                 55. Subramanian, D. & Griffith, J. D. Interactions between p53,             81. Gao, Y. et al. Interplay of p53 and DNA-repair protein
    components of the p53-mediated apoptosis pathway.                        hMSH2–hMSH6 and HMG I(Y) on Holliday junctions and                           XRCC4 in tumorigenesis, genomic stability and
    Genes Dev. 10, 1219–1232 (1996).                                         bulged bases. Nucleic Acids Res. 30, 2427–2434 (2002).                       development. Nature 404, 897–900 (2000).
31. Rubbi, C. P. & Milner, J. p53 is a chromatin accessibility           56. Lin, X. et al. p53 modulates the effect of loss of DNA                  82. Frank, K. M. et al. DNA ligase IV deficiency in mice leads to
    factor for nucleotide excision repair of DNA damage.                     mismatch repair on the sensitivity of human colon cancer                     defective neurogenesis and embryonic lethality via the p53
    EMBO J. 22, 975–986 (2003).                                              cells to the cytotoxic and mutagenic effects of cisplatin.                   pathway. Mol. Cell 5, 993–1002 (2000).
    Proposes that wild-type p53 can facilitate access of                     Cancer Res. 61, 1508–1516 (2001).                                       83. Zhu, C. et al. Unrepaired DNA breaks in p53-deficient cells
    the DNA-repair complex to the sites of DNA damage.                   57. Zink, D., Mayr, C., Janz, C. & Wiesmuller, L. Association of                 lead to oncogenic gene amplification subsequent to
32. Adimoolam, S. & Ford, J. M. p53 and regulation of DNA                    p53 and MSH2 with recombinative repair complexes during                      translocations. Cell 109, 811–821 (2002).
    damage recognition during nucleotide excision repair.                    S phase. Oncogene 21, 4788–4800 (2002).                                 84. Rooney, S. et al. Artemis and p53 cooperate to suppress
    DNA Repair (Amst.) 2, 947–954 (2003).                                58. Xinarianos, G. et al. p53 status correlates with the differential            oncogenic N-myc amplification in progenitor B cells.
33. Therrien, J. P., Drouin, R., Baril, C. & Drobetsky, E. A.                expression of the DNA mismatch repair protein MSH2 in                        Proc. Natl Acad. Sci. USA 101, 2410–2415 (2004).
    Human cells compromised for p53 function exhibit defective               non-small cell lung carcinoma. Int. J. Cancer 101, 248–252              85. Mazin, A. V., Alexeev, A. A. & Kowalczykowski, S. C. A novel
    global and transcription-coupled nucleotide excision repair,             (2002).                                                                      function of Rad54 protein. Stabilization of the Rad51
    whereas cells compromised for pRb function are defective             59. Saito, T. et al. Possible association between tumor-                         nucleoprotein filament. J. Biol. Chem. 278, 14029–14036
    only in global repair. Proc. Natl Acad. Sci. USA 96,                     suppressor gene mutations and hMSH2/hMLH1 inactivation                       (2003).
    15038–15043 (1999).                                                      in alveolar soft part sarcoma. Hum. Pathol. 34, 841–849 (2003).         86. Dudenhoffer, C., Rohaly, G., Will, K., Deppert, W. &
34. Ljungman, M. & Lane, D. P. Transcription — guarding the              60. Staibano, S. et al. p53 and hMSH2 expression in basal cell                   Wiesmuller, L. Specific mismatch recognition in
    genome by sensing DNA damage. Nature Rev. Cancer 4,                      carcinomas and malignant melanomas from photoexposed                         heteroduplex intermediates by p53 suggests a role in fidelity
    727–737 (2004).                                                          areas of head and neck region. Int. J. Oncol. 19, 551–559                    control of homologous recombination. Mol. Cell. Biol. 18,
35. Ford, J. M. & Hanawalt, P. C. Li-Fraumeni syndrome                       (2001).                                                                      5332–5342 (1998).
    fibroblasts homozygous for p53 mutations are deficient in            61. Yano, M. et al. Close correlation between a p53 or hMSH2                     The first study that indicates a fidelity-control function
    global DNA repair but exhibit normal transcription-coupled               gene mutation in the tumor and survival of hepatocellular                    of p53 in homologous recombination.
    repair and enhanced UV resistance. Proc. Natl Acad. Sci.                 carcinoma patients. Int. J. Oncol. 14, 447–451 (1999).                  87. Xia, F., Amundson, S. A., Nickoloff, J. A. & Liber, H. L.
    USA 92, 8876–8880 (1995).                                            62. Zhu, Y. M., Das-Gupta, E. P. & Russell, N. H. Microsatellite                 Different capacities for recombination in closely related
36. Adimoolam, S., Lin, C. X. & Ford, J. M. The p53-regulated                instability and p53 mutations are associated with abnormal                   human lymphoblastoid cell lines with different mutational
    cyclin-dependent kinase inhibitor, p21 (cip1, waf1, sdi1), is            expression of the MSH2 gene in adult acute leukemia.                         responses to X-irradiation. Mol. Cell. Biol. 14, 5850–5857
    not required for global genomic and transcription-coupled                Blood 94, 733–740 (1999).                                                    (1994).
    nucleotide excision repair of UV-induced DNA                         63. Leung, S. Y. et al. Chromosomal instability and p53                     88. Wiesmuller, L., Cammenga, J. & Deppert, W. W. In vivo
    photoproducts. J. Biol. Chem. 276, 25813–25822 (2001).                   inactivation are required for genesis of glioblastoma but not                assay of p53 function in homologous recombination
37. Wani, M. A., Zhu, Q., El-Mahdy, M., Venkatachalam, S. &                  for colorectal cancer in patients with germline mismatch                     between simian virus 40 chromosomes. J. Virol. 70,
    Wani, A. A. Enhanced sensitivity to anti-benzo(a)pyrene-diol-            repair gene mutation. Oncogene 19, 4079–4083 (2000).                         737–744 (1996).
    epoxide DNA damage correlates with decreased global                  64. Rothkamm, K., Kruger, I., Thompson, L. H. & Lobrich, M.                 89. Dudenhoffer, C., Kurth, M., Janus, F., Deppert, W. &
    genomic repair attributable to abrogated p53 function in                 Pathways of DNA double-strand break repair during the                        Wiesmuller, L. Dissociation of the recombination Control and
    human cells. Cancer Res. 60, 2273–2280 (2000).                           mammalian cell cycle. Mol. Cell. Biol. 23, 5706–5715                         the sequence-specific transactivation function of p53.
38. Mathonnet, G. et al. UV wavelength-dependent regulation of               (2003).                                                                      Oncogene 18, 5773–5784 (1999).
    transcription-coupled nucleotide excision repair in                  65. Shieh, S. Y., Ikeda, M., Taya, Y. & Prives, C. DNA damage-                   References 89 and 98 are key studies that showed the
    p53-deficient human cells. Proc. Natl Acad. Sci. USA 100,                induced phosphorylation of p53 alleviates inhibition by                      transactivation-independent role of p53 during the
    7219–7224 (2003).                                                        MDM2. Cell 91, 325–334 (1997).                                               modulation of HR.
39. Yu, A., Fan, H. Y., Liao, D., Bailey, A. D. & Weiner, A. M.          66. Wang, S. et al. The catalytic subunit of DNA-dependent                  90. Bertrand, P. et al. Increase of spontaneous
    Activation of p53 or loss of the Cockayne syndrome group B               protein kinase selectively regulates p53-dependent                           intrachromosomal homologous recombination in
    repair protein causes metaphase fragility of human U1, U2,               apoptosis but not cell-cycle arrest. Proc. Natl Acad. Sci.                   mammalian cells expressing a mutant p53 protein.
    and 5S genes. Mol. Cell 5, 801–810 (2000).                               USA 97, 1584–1588 (2000).                                                    Oncogene 14, 1117–1122 (1997).
40. Gaiddon, C., Moorthy, N. C. & Prives, C. Ref-1 regulates the         67. Jhappan, C., Yusufzai, T. M., Anderson, S., Anver, M. R. &              91. Saintigny, Y. & Lopez, B. S. Homologous recombination
    transactivation and pro-apoptotic functions of p53 in vivo.              Merlino, G. The p53 response to DNA damage in vivo is                        induced by replication inhibition, is stimulated by expression
    EMBO J. 18, 5609–5621 (1999).                                            independent of DNA-dependent protein kinase. Mol. Cell.                      of mutant p53. Oncogene 21, 488–492 (2002).
41. Offer, H. et al. Direct involvement of p53 in the base excision          Biol. 20, 4075–4083 (2000).                                             92. Mekeel, K. L. et al. Inactivation of p53 results in high rates of
    repair pathway of the DNA repair machinery. FEBS Lett.               68. Jimenez, G. S. et al. DNA-dependent protein kinase is not                    homologous recombination. Oncogene 14, 1847–1857
    450, 197–204 (1999).                                                     required for the p53-dependent response to DNA damage.                       (1997).
    The initial observation of BER modulation by wild-type                   Nature 400, 81–83 (1999).                                               93. Saintigny, Y., Rouillard, D., Chaput, B., Soussi, T. &
    p53.                                                                 69. Achanta, G., Pelicano, H., Feng, L., Plunkett, W. & Huang, P.                Lopez, B. S. Mutant p53 proteins stimulate spontaneous
42. Offer, H. et al. The onset of p53-dependent DNA repair or                Interaction of p53 and DNA-PK in response to nucleoside                      and radiation-induced intrachromosomal homologous
    apoptosis is determined by the level of accumulated                      analogues: potential role as a sensor complex for DNA                        recombination independently of the alteration of the
    damaged DNA. Carcinogenesis 23, 1025–1032 (2002).                        damage. Cancer Res. 61, 8723–8729 (2001).                                    transactivation activity and of the G1 checkpoint. Oncogene
43. Offer, H. et al. p53 modulates base excision repair activity in      70. Yang, T. et al. p53 induced by ionizing radiation mediates                   18, 3553–3563 (1999).
    a cell cycle-specific manner after genotoxic stress. Cancer              DNA end-jointing activity, but not apoptosis of thyroid cells.          94. Bishop, A. J. et al. Atm-, p53-, and Gadd45a-deficient mice
    Res. 61, 88–96 (2001).                                                   Oncogene 14, 1511–1519 (1997).                                               show an increased frequency of homologous recombination
44. Zurer, I. et al. The role of p53 in base excision repair following   71. Tang, W., Willers, H. & Powell, S. N. p53 directly enhances                  at different stages during development. Cancer Res. 63,
    genotoxic stress. Carcinogenesis 25, 11–19 (2004).                       rejoining of DNA double-strand breaks with cohesive ends in                  5335–5343 (2003).
45. Achanta, G. & Huang, P. Role of p53 in sensing oxidative                 γ-irradiated mouse fibroblasts. Cancer Res. 59, 2562–2565               95. Janz, C., Susse, S. & Wiesmuller, L. p53 and recombination
    DNA damage in response to reactive oxygen species-                       (1999).                                                                      intermediates: role of tetramerization at DNA junctions in
    generating agents. Cancer Res. 64, 6233–6239 (2004).                 72. Lin, Y., Waldman, B. C. & Waldman, A. S. Suppression of                      complex formation and exonucleolytic degradation.
46. Sobol, R. W. et al. Base excision repair intermediates induce            high-fidelity double-strand break repair in mammalian                        Oncogene 21, 2130–2140 (2002).
    p53-independent cytotoxic and genotoxic responses.                       chromosomes by pifithrin-α, a chemical inhibitor of p53.                96. Lin, J., Chen, J., Elenbaas, B. & Levine, A. J. Several
    J. Biol. Chem. 278, 39951–39959 (2003).                                  DNA Repair (Amst.) 2, 1–11 (2003).                                           hydrophobic amino acids in the p53 amino-terminal domain
47. Zhou, J., Ahn, J., Wilson, S. H. & Prives, C. A role for p53 in      73. Bristow, R. G. et al. Radioresistant MTp53-expressing rat                    are required for transcriptional activation, binding to mdm-2
    base excision repair. EMBO J. 20, 914–923 (2001).                        embryo cell transformants exhibit increased DNA-dsb                          and the adenovirus 5 E1B 55-kD protein. Genes Dev. 8,
    References 44 and 47 show the in vivo involvement of                     rejoining during exposure to ionizing radiation. Oncogene                    1235–1246 (1994).
    p53 in BER.                                                              16, 1789–1802 (1998).                                                   97. Sengupta, S. et al. BLM helicase-dependent transport of
48. Seo, Y. R., Fishel, M. L., Amundson, S., Kelley, M. R. &             74. Bill, C. A., Yu, Y., Miselis, N. R., Little, J. B. & Nickoloff, J. A.        p53 to sites of stalled DNA replication forks modulates
    Smith, M. L. Implication of p53 in base excision DNA repair:             A role for p53 in DNA end rejoining by human cell extracts.                  homologous recombination. EMBO J. 22, 1210–1222
    in vivo evidence. Oncogene 21, 731–737 (2002).                           Mutat. Res. 385, 21–29 (1997).                                               (2003).
49. Offer, H. et al. Structural and functional involvement of p53 in     75. Akyuz, N. et al. DNA substrate dependence of p53-                            References 97, 113 and 115 indicate the functional
    BER in vitro and in vivo. Oncogene 20, 581–589 (2001).                   mediated regulation of double-strand break repair. Mol. Cell.                relationship between wild-type p53 and BLM helicase.
50. Seo, Y. R., Kelley, M. R. & Smith, M. L. Selenomethionine                Biol. 22, 6306–6317 (2002).                                             98. Willers, H. et al. Dissociation of p53-mediated suppression
    regulation of p53 by a ref1-dependent redox mechanism.               76. Lee, H., Sun, D., Larner, J. M. & Wu, F. S. The tumor                        of homologous recombination from G1/S cell cycle
    Proc. Natl Acad. Sci. USA 99, 14548–14553 (2002).                        suppressor p53 can reduce stable transfection in the                         checkpoint control. Oncogene 19, 632–639 (2000).
51. Peltomaki, P. Role of DNA mismatch repair defects in the                 presence of irradiation. J. Biomed. Sci. 6, 285–292 (1999).             99. Boehden, G. S., Akyuz, N., Roemer, K. & Wiesmuller, L.
    pathogenesis of human cancer. J. Clin. Oncol. 21,                    77. Okorokov, A. L., Warnock, L. & Milner, J. Effect of wild-type,               p53 mutated in the transactivation domain retains regulatory
    1174–1179 (2003).                                                        S15D and R175H p53 proteins on DNA end joining in vitro:                     functions in homology-directed double-strand break repair.
52. Luo, Y., Lin, F. T. & Lin, W. C. ATM-mediated stabilization of           potential mechanism of DNA double-strand break repair                        Oncogene 22, 4111–4117 (2003).
    hMutL DNA mismatch repair proteins augments p53                          modulation. Carcinogenesis 23, 549–557 (2002).                          100. Linke, S. P. et al. p53 interacts with hRAD51 and hRAD54,
    activation during DNA damage. Mol. Cell. Biol. 24,                   78. Gu, Y. et al. Growth retardation and leaky SCID phenotype                    and directly modulates homologous recombination. Cancer
    6430–6444 (2004).                                                        of Ku70-deficient mice. Immunity 7, 653–665 (1997).                          Res. 63, 2596–2605 (2003).
53. Cranston, A. et al. Female embryonic lethality in mice               79. Frank, K. M. et al. Late embryonic lethality and impaired               101. Sturzbecher, H. W., Donzelmann, B., Henning, W.,
    nullizygous for both Msh2 and p53. Nature Genet. 17,                     V(D)J recombination in mice lacking DNA ligase IV. Nature                    Knippschild, U. & Buchhop, S. p53 is linked directly to
    114–118 (1997).                                                          396, 173–177 (1998).                                                         homologous recombination processes via RAD51/RecA
54. Scherer, S. J. et al. p53 and c-Jun functionally synergize in        80. Gao, Y. et al. A critical role for DNA end-joining proteins in               protein interaction. EMBO J. 15, 1992–2002 (1996).
    the regulation of the DNA repair gene hMSH2 in response to               both lymphogenesis and neurogenesis. Cell 95, 891–902                   102. Buchhop, S. et al. Interaction of p53 with the human Rad51
    UV. J. Biol. Chem. 275, 37469–37473 (2000).                              (1998).                                                                      protein. Nucleic Acids Res. 25, 3868–3874 (1997).

54   | JANUARY 2005 | VOLUME 6                                                                                                                                

103. Susse, S., Janz, C., Janus, F., Deppert, W. & Wiesmuller, L.               co-localization with other nuclear proteins. Cytogenet. Cell    137. Ward, I. M. & Chen, J. Histone H2AX is phosphorylated in an
     Role of heteroduplex joints in the functional interactions                 Genet. 91, 217–223 (2000).                                           ATR-dependent manner in response to replicational stress.
     between human Rad51 and wild-type p53. Oncogene 19,                 120.   Yankiwski, V., Marciniak, R. A., Guarente, L. & Neff, N. F.          J. Biol. Chem. 276, 47759–47762 (2001).
     4500–4512 (2000).                                                          Nuclear structure in normal and Bloom syndrome cells.           138. Davies, S. L., North, P. S., Dart, A., Lakin, N. D. &
104. Yoon, Y., Wang, Y., Stapleford, K., Wiesmuller, L. & Chen, C.              Proc. Natl Acad. Sci. USA 97, 5214–5219 (2000).                      Hickson, I. D. Phosphorylation of the Bloom’s syndrome
     p53 inhibits strand exchange and replication fork regression        121.   Maacke, H. et al. DNA repair and recombination factor                helicase and its role in recovery from S-phase arrest.
     promoted by Rad51. J. Mol. Biol. 336, 639–654 (2004).                      Rad51 is over-expressed in human pancreatic                          Mol. Cell. Biol. 24, 1279–1291 (2004).
105. Willers, H., McCarthy, E. E., Hubbe, P., Dahm-Daphi, J. &                  adenocarcinoma. Oncogene 19, 2791–2795 (2000).                  139. Sengupta, S. et al. Functional interaction between
     Powell, S. N. Homologous recombination in                           122.   Xia, S. J., Shammas, M. A. & Shmookler Reis, R. J. Elevated          BLM helicase and 53BP1 in a Chk1-mediated
     extrachromosomal plasmid substrates is not suppressed by                   recombination in immortal human cells is mediated by                 pathway during S-phase arrest. J. Cell Biol. 166, 801–813
     p53. Carcinogenesis 22, 1757–1763 (2001).                                  HsRAD51 recombinase. Mol. Cell. Biol. 17, 7151–7158 (1997).          (2004).
106. Kumari, A., Schultz, N. & Helleday, T. p53 protects from            123.   Lebel, M. & Leder, P. A deletion within the murine Werner       140. Tibbetts, R. S. et al. A role for ATR in the DNA damage-
     replication-associated DNA double-strand breaks in                         syndrome helicase induces sensitivity to inhibitors of               induced phosphorylation of p53. Genes Dev. 13, 152–157
     mammalian cells. Oncogene 23, 2324–2329 (2004).                            topoisomerase and loss of cellular proliferative capacity.           (1999).
107. Hickson, I. D. RecQ helicases: caretakers of the genome.                   Proc. Natl Acad. Sci. USA 95, 13097–13102 (1998).               141. Shieh, S. Y., Ahn, J., Tamai, K., Taya, Y. & Prives, C.
     Nature Rev. Cancer 3, 169–178 (2003).                               124.   Lebel, M., Cardiff, R. D. & Leder, P. Tumorigenic effect of          The human homologs of checkpoint kinases Chk1
108. Wu, L. & Hickson, I. D. The Bloom’s syndrome helicase                      nonfunctional p53 or p21 in mice mutant in the Werner                and Cds1 (Chk2) phosphorylate p53 at multiple
     suppresses crossing over during homologous                                 syndrome helicase. Cancer Res. 61, 1816–1819 (2001).                 DNA damage-inducible sites. Genes Dev. 14, 289–300
     recombination. Nature 426, 870–874 (2003).                          125.   Chang, S. et al. Essential role of limiting telomeres in the         (2000).
109. Saintigny, Y., Makienko, K., Swanson, C., Emond, M. J. &                   pathogenesis of Werner syndrome. Nature Genet. 36,              142. Gottifredi, V., Shieh, S., Taya, Y. & Prives, C. p53
     Monnat, R. J. Jr. Homologous recombination resolution                      877–882 (2004).                                                      accumulates but is functionally impaired when DNA
     defect in werner syndrome. Mol. Cell. Biol. 22, 6971–6978           126.   Fukasawa, K., Choi, T., Kuriyama, R., Rulong, S. &                   synthesis is blocked. Proc. Natl Acad. Sci. USA 98,
     (2002).                                                                    Vande Woude, G. F. Abnormal centrosome amplification in              1036–1041 (2001).
110. Yamabe, Y. et al. Sp1-mediated transcription of the Werner                 the absence of p53. Science 271, 1744–1747 (1996).                   An important study that suggests the S-phase
     helicase gene is modulated by Rb and p53. Mol. Cell. Biol.          127.   Cross, S. M. et al. A p53-dependent mouse spindle                    accumulation of transactivation-deficient p53.
     18, 6191–6200 (1998).                                                      checkpoint. Science 267, 1353–1356 (1995).                      143. Lane, D. P. p53, guardian of the genome. Nature 358,
111. Garkavtsev, I. V., Kley, N., Grigorian, I. A. & Gudkov, A. V. The          References 126 and 127 show the effect of mutant p53                 15–16 (1992).
     Bloom syndrome protein interacts and cooperates with p53                   on chromosomal aberrations.                                     144. Baptiste, N. & Prives, C. p53 in the cytoplasm: a question of
     in regulation of transcription and cell growth control.             128.   Bouffler, S. D., Kemp, C. J., Balmain, A. & Cox, R.                  overkill? Cell 116, 487–489 (2004).
     Oncogene 20, 8276–8280 (2001).                                             Spontaneous and ionizing radiation-induced chromosomal
112. Spillare, E. A. et al. p53-mediated apoptosis is attenuated in             abnormalities in p53-deficient mice. Cancer Res. 55,            Acknowledgments
     Werner syndrome cells. Genes Dev. 13, 1355–1360 (1999).                    3883–3889 (1995).                                               We thank J. Bradsher, X. Wang, J. Shen and S. Linke for helpful
113. Wang, X. W. et al. Functional interaction of p53 and BLM            129.   Bunz, F. et al. Targeted inactivation of p53 in human cells     discussions. We also thank D. Dudek for editorial assistance and
     DNA helicase in apoptosis. J. Biol. Chem. 276,                             does not result in aneuploidy. Cancer Res. 62, 1129–1133        K. MacPherson for bibliographic assistance.
     32948–32955 (2001).                                                        (2002).
114. Blander, G. et al. Physical and functional interaction between             Argues that the inactivation of wild-type p53 does not          Competing interests statement
     p53 and the Werner’s syndrome protein. J. Biol. Chem. 274,                 result in aneuploidy.                                           The authors declare no competing financial interests.
     29463–29469 (1999).                                                 130.   Shiloh, Y. ATM: ready, set, go. Cell Cycle 2, 116–117 (2003).
115. Yang, Q. et al. The processing of Holliday junctions by BLM         131.   Abraham, R. T. Cell cycle checkpoint signaling through the
     and WRN helicases is regulated by p53. J. Biol. Chem. 277,                 ATM and ATR kinases. Genes Dev. 15, 2177–2196 (2001).                  Online links
     31980–31987 (2002).                                                 132.   Bartek, J. & Lukas, J. Chk1 and Chk2 kinases in checkpoint
116. Blander, G. et al. The Werner syndrome protein contributes                 control and cancer. Cancer Cell 3, 421–429 (2003).              DATABASES
     to induction of p53 by DNA damage. FASEB J. 14,                     133.   Rogakou, E. P., Boon, C., Redon, C. & Bonner, W. M.             The following terms in this article are linked online to:
     2138–2140 (2000).                                                          Megabase chromatin domains involved in DNA double-              Entrez:
117. Brosh, R. M. Jr. et al. p53 modulates the exonuclease                      strand breaks in vivo. J. Cell Biol. 146, 905–916 (1999).       BLM | RECQ4 | Terc | WRN
     activity of Werner syndrome protein. J. Biol. Chem. 276,            134.   Motoyama, N. & Naka, K. DNA damage tumor suppressor             OMIM:
     35093–35102 (2001).                                                        genes and genomic instability. Curr. Opin. Genet. Dev. 14,      Bloom syndrome | HNPCC | Rothmund–Thomson syndrome |
118. Waterman, M. J., Stavridi, E. S., Waterman, J. L. &                        11–16 (2004).                                                   Werner syndrome | Xeroderma pigmentosum
     Halazonetis, T. D. ATM-dependent activation of p53 involves         135.   Ahn, J., Urist, M. & Prives, C. Questioning the role of         Swiss-Prot:
     dephosphorylation and association with 14-3-3 proteins.                    checkpoint kinase 2 in the p53 DNA damage response.             APE1 | ATM | ATR | ATRIP | CHK2 | CSA | CSB | DNA ligase IV |
     Nature Genet. 19, 175–178 (1998).                                          J. Biol. Chem. 278, 20480–20489 (2003).                         Jun | Ku70 | Ku80 | MDM2 | MLH1 | MSH2 | MSH6 | p48DDB2 | p53
119. Sanz, M. M., Proytcheva, M., Ellis, N. A., Holloman, W. K. &        136.   Zou, L. & Elledge, S. J. Sensing DNA damage through             | p127DDB1 | PCNA | PMS2 | RAD23B | RAD51 | RAD52 | Sp1 |
     German, J. BLM, the Bloom’s syndrome protein, varies                       ATRIP recognition of RPA–ssDNA complexes. Science 300,          XPA | XPB | XPC | XPD | XPF | XRCC4
     during the cell cycle in its amount, distribution, and                     1542–1548 (2003).                                               Access to this links box is available online.

NATURE REVIEWS | MOLECUL AR CELL BIOLOGY                                                                                                                              VOLUME 6 | JANUARY 2005 | 5 5

Shared By: