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					                  Biological repair mechanisms
   There are many potential threats to the fidelity of DNA replication.
    Not only is there an inherent error rate for the replication of DNA, but
    there are also spontaneous lesions that can provoke additional errors.
    Moreover, mutagens in the environment can damage DNA and greatly
    increase the mutation rate.
   Living cells have evolved a series of enzymatic systems that repair
    DNA damage in a variety of ways. Failure of these systems can lead
    to a higher mutation rate. A number of human diseases including
    certain types of cancer can be attributed to defects in DNA repair, as
    we shall see later. Let's first examine some of the characterized repair
    pathways and then consider how the cell integrates these systems into
    an overall strategy for repair.
   We can divide repair pathways into several categories.

                                                      Genetica per Scienze Naturali
                                                      a.a. 05-06 prof S. Presciuttini
          Prevention of errors before they happen
   Some enzymatic systems neutralize potentially damaging compounds
    before they even react with DNA. One example of such a system is
    the detoxification of superoxide radicals produced during oxidative
    damage to DNA: the enzyme superoxide dismutase catalyzes the
    conversion of the superoxide radicals into hydrogen peroxide, and the
    enzyme catalase, in turn, converts the hydrogen peroxide into water.
    Another error-prevention pathway depends on the protein product of
    the mutT gene: this enzyme prevents the incorporation of 8-oxodG,
    which arises by oxidation of dGTP, into DNA by hydrolyzing the
    triphosphate of 8-oxodG back to the monophosphate

                                   DNA damage products formed after attack by
                                   oxygen radicals. dR = deoxyribose



                                                      Genetica per Scienze Naturali
                                                      a.a. 05-06 prof S. Presciuttini
                       Direct reversal of damage
   The most straightforward way to repair a lesion, once it occurs, is to reverse it
    directly, thereby regenerating the normal base. Reversal is not always possible,
    because some types of damage are essentially irreversible. In a few cases, however,
    lesions can be repaired in this way. One case is a mutagenic photodimer caused by
    UV light. The cyclobutane pyrimidine photodimer can be repaired by a photolyase
    that has been found in bacteria and lower eukaryotes but not in humans. The enzyme
    binds to the photodimer and splits it, in the presence of certain wavelengths of
    visible light, to generate the original bases. This enzyme cannot operate in the dark,
    so other repair pathways are required to remove UV damage. A photolyase that
    reverses the 6-4 photoproducts has been detected in plants and Drosophila.

                                 Repair of a UV-induced pyrimidine photodimer by a
                                 photoreactivating enzyme, or photolyase. The enzyme
                                 recognizes the photodimer (here, a thymine dimer) and
                                 binds to it. When light is present, the photolyase uses
                                 its energy to split the dimer into the original monomers.

                                                                Genetica per Scienze Naturali
                                                                a.a. 05-06 prof S. Presciuttini
                        Removal of alkyl groups
   Alkyltransferases also are enzymes taking part in the direct reversal of lesions.
    They remove certain alkyl groups that have been added to the O-6 positions of
    guanine by such agents as NG and EMS. The methyltransferase from E. coli has
    been well studied. This enzyme transfers the methyl group from O-6-methylguanine
    to a cysteine residue on the protein. When this happens, the enzyme is inactivated,
    so this repair system can be saturated if the level of alkylation is high enough.



                                    Alkylation-induced specific mispairing. The
                                    alkylation (in this case, EMS-generated ethylation)
                                    of the O-6 position of guanine and the O-4 position
                                    of thymine can lead to direct mispairing with
                                    thymine and guanine, respectively, as shown here.
                                    In bacteria, where mutations have been analyzed in
                                    great detail, the principal mutations detected are
                                    GC → AT transitions, indicating that the O-6
                                    alkylation of guanine is most relevant to
                                    mutagenesis.                Genetica per Scienze Naturali
                                                                a.a. 05-06 prof S. Presciuttini
Excision-repair pathways
              Also termed nucleotide excision
              repair, this system includes the
              breaking of a phosphodiester
              bond on either side of the lesion,
              on the same strand, resulting in
              the excision of an
              oligonucleotide. This excision
              leaves a gap that is filled by
              repair synthesis, and a ligase
              seals the breaks. In prokaryotes,
              12 or 13 nucleotides are
              removed; whereas, in
              eukaryotes, from 27 to 29
              nucleotides are eliminated.

                          Genetica per Scienze Naturali
                          a.a. 05-06 prof S. Presciuttini
                        The excinuclease
   In E. coli, the products of the uvrA, B, and C genes constitute the
    excinuclease. The UvrA protein, which recognizes the damaged DNA,
    forms a complex with UvrB and leads the UvrB subunit to the damage
    site before dissociating. The UvrC protein then binds to UvrB. Each
    of these subunits makes an incision. The short DNA 12-mer is
    unwound and released by another protein, helicase II.
                            The human excinuclease is considerably
                            more complex than its bacterial counterpart
                            and includes at least 17 proteins. However,
                            the basic steps are the same as those in E.
                            coli

                          Schematic representation of events following incision
                          by UvrABC exinuclease in E. coli.


                                                       Genetica per Scienze Naturali
                                                       a.a. 05-06 prof S. Presciuttini
                 Excision repair defects in humans
   Several human genetic diseases are known to be due to repair defects.
   Xeroderma pigmentosum (XP) results from a defect in any of the
    genes (complementation groups) effecting nucleotide excision repair.
    People suffering from this disorder are extremely prone to UVinduced
    skin cancers as a result of exposure to sunlight and have frequent
    neurological abnormalities.
Nucleotide excision repair is coupled to transcription. This model for coupled repair in
mammalian cells shows RNA polymerase (pink) pausing when encountering a lesion. It
undergoes a conformational change, allowing the DNA strands at the lesion site to
reanneal.
                                                         Protein factors aid in coupling
                                                         by bringing TFIIH and other
                                                         factors to the site to carry out
                                                         the incision, excision, and repair
                                                         reactions. Then transcription can
                                                         continue normally.
                                                                Genetica per Scienze Naturali
                                                                a.a. 05-06 prof S. Presciuttini
Specific excision pathways
      Certain lesions are too subtle to cause a distortion
       large enough to be recognized by the uvrABC-
       encoded general excision-repair system and its
       counterparts in higher cells. Thus, additional
       excision pathways are necessary.
      DNA glycosylase repair pathway (base-excision
       repair). DNA glycosylases do not cleave
       phosphodiester bonds, but instead cleave N-
       glycosidic (base–sugar) bonds, liberating the
       altered base and generating an apurinic or an
       apyrimidinic site, both called AP sites, because
       they are biochemically equivalent. The resulting
       Ap site is then repaired by an AP endonuclease
       repair pathway


                                    Genetica per Scienze Naturali
                                    a.a. 05-06 prof S. Presciuttini
                              Mismatch repair
   Some repair pathways are capable of recognizing errors even after
    DNA replication has already occurred. One such system, termed the
    mismatch repair system, can detect mismatches that occur in DNA
    replication. Suppose you were to design an enzyme system that could
    repair replication errors. What would this system have to be able to
    do? At least three things:
       1. Recognize mismatched base pairs.
       2. Determine which base in the mismatch is the incorrect one.
       3. Excise the incorrect base and carry out repair synthesis.
   The second point is the crucial property of such a system. Unless it is
    capable of discriminating between the correct and the incorrect bases,
    the mismatch repair system could not determine which base to excise.
    If, for example, a G–T mismatch occurs as a replication error, how
    can the system determine whether G or T is incorrect? Both are
    normal bases in DNA. But replication errors produce mismatches on
    the newly synthesized strand, so it is the base on this strand that must
    be recognized and excised.
                                                             Genetica per Scienze Naturali
                                                             a.a. 05-06 prof S. Presciuttini
                          DNA methylation
   To distinguish the old, template strand from the newly synthesized
    strand, the mismatch repair system in bacteria takes advantage of the
    normal delay in the postreplication methylation of the sequence




    The methylating enzyme is adenine methylase, which creates 6-
    methyladenine on each strand. However, it takes the adenine
    methylase several minutes to recognize and modify the newly
    synthesized GATC stretches. During that interval, the mismatch repair
    system can operate because it can now distinguish the old strand from
    the new one by the methylation pattern. Methylating the 6-position of
    adenine does not affect base pairing, and it provides a convenient tag
    that can be detected by other enzyme systems.
                                                     Genetica per Scienze Naturali
                                                     a.a. 05-06 prof S. Presciuttini
Mismatch repair
        Model for mismatch repair in E. coli.
        Because DNA is methylated by
        enzymatic reactions that recognize the A
        in a GATC sequence, the newly
        synthesized strand will not be methylated
        directly after DNA replication.
        The hemimethylated DNA duplex serves
        as a recognition point for the mismatch
        repair system in discerning the old from
        the new strand. Here a G–T mismatch is
        shown. The mismatch repair system can
        recognize and bind to this mismatch,
        determine the correct (old) strand
        because it is the methylated strand of a
        hemimethylated duplex, and then excise
        the mismatched base from the new
        strand. Repair synthesis restores the
        normal base pair.

                       Genetica per Scienze Naturali
                       a.a. 05-06 prof S. Presciuttini
The complex MutS-MutH
       Steps in E. coli mismatch repair.
           (1) MutS binds to mispair.
           (2) MutH and MutL are recruited to form a
            complex. MutH cuts the newly synthesized
            (unmethylated) strand, and exonuclease
            degradation goes past the point of the mismatch,
            leaving a patch.
           (3) Single-strand-binding protein (Ssb) protects
            the single-stranded region across from the
            missing patch.
           (4) Repair synthesis and ligation fill in the gap.




                                    Genetica per Scienze Naturali
                                    a.a. 05-06 prof S. Presciuttini
Mismatch repair defects in humans
     Hereditary nonpolyposis colorectal cancer (HNPCC) is
     one of the most common inherited predispositions to cancer,
     affecting as many as 1 in 500 people in the Western world.
     Most HNPCC results from a defect in genes that encode the
     human counterparts (and homologs) of the bacterial MutS and
     MutL proteins. The inheritance of HNPCC is autosomal
     dominant. Cells with one functional copy of the mismatch
     repair genes have normal mismatch repair activity, but tumor
     cell lines arise from cells that have lost the one functional
     copy and are thus mismatch repair deficient. These cells
     display high mutation rates that eventually result in tumor
     growth and proliferation.
     Mismatch repair in humans. (1) Mispairs and misaligned bases
     arise in the course of replication. (2) The G–T-binding protein
     (GTBP) and the human MutS homolog (hMSH2) recognize the
     incorrect matches. (3) Two additional proteins, hPMS2 and
     hMLH1, are recruited and form a larger repair complex. (4) The
     mismatch is repaired after removal, DNA synthesis, and ligation.
                                            Genetica per Scienze Naturali
                                            a.a. 05-06 prof S. Presciuttini
                                    SOS repair
DNA damage often results ina replication block, because DNA synthesis will not
proceed past a base that cannot specify its complementary partner by hydrogen
bonding. In bacterial cells, such replication blocks can be bypassed by inserting
nonspecific bases. The process requires the activation of a special system, the SOS
system. The name SOS comes from the idea that this system is induced as an
emergency response to prevent cell death in the presence of significant DNA damage.
SOS induction is a last resort, allowing the cell to trade death for a certain level of
mutagenesis.
                                          The recA gene, takes part in postreplication
                                          repair. Here the DNA replication system stalls at
                                          a UV photodimer and then restarts past the
                                          block, leaving a single-stranded gap. This
                                          process leads to few errors.
                                          SOS bypass, in contrast, is highly mutagenic.
                                          Here the replication system continues past the
                                          lesion, accepting noncomplementary
                                          nucleotides for new strand synthesis

                                                                Genetica per Scienze Naturali
                                                                a.a. 05-06 prof S. Presciuttini
                    Schemes for postreplication repair
(a) In recombinational repair, replication jumps across a blocking lesion, leaving a gap
in the new strand. A recA-directed protein then fills the gap, using a piece from the
opposite parental strand (because of DNA complemen-tarity, this filler will supply the
correct bases for the gap). Finally, the RecA protein repairs the gap in the parental
strand.
                                                          (b) In SOS bypass, when
                                                          replication reaches a blocking
                                                          lesion, the SOS system inserts
                                                          the necessary number of bases
                                                          (often incorrect ones) directly
                                                          across from the lesion and
                                                          replication continues without a
                                                          gap. Note that with either
                                                          pathway the original blocking
                                                          lesion is still there and must be
                                                          repaired by some other repair
                                                          pathway.

                                                                 Genetica per Scienze Naturali
                                                                 a.a. 05-06 prof S. Presciuttini
                 Summary of repair mechanisms
   We can now assess the overall repair system strategy used by the cell. It
    would be convenient if enzymes could be used to directly reverse each
    specific lesion. However, sometimes that is not chemically possible, and not
    every possible type of DNA damage can be anticipated.
   Therefore, a general excision repair system is used to remove any type of
    damaged base that causes a recognizable distortion in the double helix.
   When lesions are too subtle to cause such a distortion, specific excision
    systems, glycosylases, or removal systems are designed.
   To eliminate replication errors, a postreplication mismatch repair system
    operates; finally, postreplication recombinational systems eliminate gaps
    across from blocking lesions that have escaped the other repair systems.
   The SOS system is the last resort for a cell to survive a potentially lethald
    DNA damage



                                                          Genetica per Scienze Naturali
                                                          a.a. 05-06 prof S. Presciuttini
                     DNA repair and mutation rates
   The repair processes are so efficient that the observed base substitution rate is as low
    as 10−10 to 10−9 per base pair per cell per generation in E. coli. However, mutant
    strains with increased spontaneous mutation rates have been detected. Such strains
    are termed mutators. In many cases, the mutator phenotype is due to a defective
    repair system. In humans, these repair defects often lead to serious diseases.
   In E. coli, the mutator loci mutH, mutL, mutU, and mutS affect components of the
    postreplication mismatch repair system, as does the dam locus, which specifies the
    enzyme deoxyadenosine methylase. Strains that are Dam− cannot methylate
    adenines at GATC sequences, and so the mismatch repair system can no longer
    discriminate between the template and the newly synthesized strands. This failure to
    discriminate leads to a higher spontaneous mutation rate.
   Mutations in the mutY locus result in GC → TA transversions, because many G–A
    mispairs and all 8-oxodG–A mispairs are unrepaired. The mutM gene encodes a
    glycosylase that removes 8-oxodG. Strains lacking mutM are mutators for the GC →
    TA transversion. Strains that are MutT− have elevated rates of the AT → CG
    transversion, because they lack an activity that prevents the incorporation of 8-
    oxodG across from adenine.
   Strains that are Ung− are missing the enzyme uracil DNA glycosylase. These
    mutants cannot excise the uracil resulting from cytosine deaminations and, as a
    result, have elevated levels of C → T transitions. The mutD locus is responsible for
    a very high rate of mutagenesis (at least three orders of magnitude higher than
    normal). Mutations at this locus affect the proofreading functions of DNA
    polymerase III.                                                Genetica per Scienze Naturali
                                                                    a.a. 05-06 prof S. Presciuttini

				
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