Mutation-DNA Repair-2009

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					                      Endogenous DNA Damage

from Marnett and Plastaras, Trends Genet. 17, 214 (2001)
                       Biological Molecules are Labile

RNA is susceptible to hydrolysis

Reduction of ribose to deoxyribose gives DNA greater stability

N-glycosyl bond of DNA is more labile

DNA damage occurs from normal cellular operations
and random interactions with the environment
Spontaneous Changes that Alter DNA Structure



     from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-46
                Hydrolysis of the N-glycosyl Bond of DNA

                   from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-47

Spontaneous depurination results in loss of 10,000 bases/cell/day

Causes formation of an AP site – not mutagenic
                  Deamination of Cytosine to Uracil

                   from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-47

Cytosine is deaminated to uracil at a rate of 100-500/cell/day

Uracil is excised by uracil-DNA-glycosylase to form AP site
          5-Methyl Cytosine Deamination is Highly Mutagenic

                                                                        Deamination of 5-methyl
                                                                        cytosine to T occurs rapidly
                                                                        - base pairs with A

                                                                        5-me-C is a target for
                                                                        spontaneous mutations

from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-52
     Deamination of A and G Occur Less Frequently

                                                A is deaminated to HX – base pairs with C

                                                G is deaminated to X – base pairs with C

from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-52
                        Oxidative Damage of DNA

Oxidative damage results from aerobic metabolism, environmental toxins,
activated macrophages, and signaling molecules (NO)

Compartmentation limits oxidative DNA damage
     Oxidation of Guanine Forms 8-Oxoguanine

                                               The most common mutagenic
                                               base lesion is 8-oxoguanine

 guanine                      8-oxoguanine

from Banerjee et al., Nature 434, 612 (2005)
Repair of 8-oxoG

                   Replication of the 8-oxoG strand
                   preferentially mispairs with A
                   and mimics a normal base pair
                   and results in a G-to-T transversion

                   8-oxoguanine DNA glycosylase/
                   b-lyase (OGG1) removes 8-oxo-G
                   and creates an AP site

                   MUTYH removes the A opposite 8-oxoG
               Oxidation of dNTPs are Mutagenic

cGTP is oxidized to 8-OH-dGTP and is misincorporated opposite A

MutT converts 8-OH-dGTP to 8-OH-dGMP
UV-Irradiation Causes Formation of Thymine Dimers

         from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-38
                  Nonenzymatic Methylation of DNA

Formation of 600 3-me-A residues/cell/day are caused by S-andnosylmethionine

3-me-A is cytotoxic and is repaired by 3-me-A-DNA glycosylase

7-me-G is the main aberrant base present in DNA and
is repaired by nonenzymatic cleavage of the glycosyl bond
                       Effect of Chemical Mutagens

Nitrous acid causes deamination of C to U and A to HX

U base pairs with A
HX base pairs with C
  Spores Use Strategies to Overcome Intrinsic Instability of DNA

DNA exists in an A-like conformation and is bound
to proteins that reduce the rate of depurination

Lack of dNTPs in spores prevents DNA repair before germination

Extensive DNA repair occurs upon spore germination
    Repair Pathways for Altered DNA Bases

from Lindahl and Wood, Science 286, 1897 (1999)
                             Direct Repair of DNA

Photoreactivation of pyrimidine dimers by photolyase restores the original DNA structure

O6-methylguanine is repaired by removal of methyl group by MGMT

1-methyladenine and 3-methylcytosine are repaired by oxidative demethylation
            Base Excision Repair of a G-T Mismatch

                                                         BER works primarily on modifications
                                                         caused by endogenous agents

                                                         At least 8 DNA glcosylases are
                                                         present in mammalian cells

                                                         DNA glycosylases remove
                                                         mismatched or abnormal bases

                                                         AP endonuclease cleaves 5’ to AP site

                                                         AP lyase cleaves 3’ to AP site

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-36
                   Mechanism of hOGG1 Action

                                                from David, Nature 434, 569 (2005)

hOGG1 binds nonspecifically to DNA

Contacts with C results in the extrusion of corresponding base in the opposite strand

G is extruded into the G-specific pocket,
but is denied access to the oxoG pocket

oxoG moves out of the G-specific pocket, enters the
oxoG-specific pocket, and excised from the DNA
Nucleotide Excision Repair in Human Cells

                          NER works mainly on helix-distorting
                          damage caused by environmental mutagens

                          The only pathway to repair thymine dimers
                          in humans is nucleotide excision repair

                          Mutations in at least seven XP genes
                          inactivate nucleotide excision repair
                          and cause xeroderma pigmentosum

                          XPC recognizes damaged DNA

                          Helicase activities of XPB and XPD of
                          TFIIH create sites for XPF and XPG cleavage

                          An oligonucleotide containing the
                          lesion is released and the gap is filled
                          by POL d or e and sealed by LIG1

     from Lindahl and Wood, Science 286, 1897 (1999)
                      Transcription-coupled Repair

Repair of the transcribed strand of active genes is
corrected 5-10-fold as fast as the nontranscribed strand

All the factors required for NER are required for transcription-coupled repair except XPC

The arrest of POL II progression at a lesion served as a damage recognition signal

Recruitment of NER factors also involves CS-A and CS-B
         Nucleotide Excision Repair Pathway in Mammals

Cockayne’s Syndrome and Trichothiodystrophy
are multisystem disorders defective in
transcription-coupled DNA repair
Mismatch Repair in E. coli

                 Newly replicated DNA is hemimethylated

                 MutS binds to mismatch and recruits MutL

                 Activates endonuclease activity of MutH
                 and nicks the nearest unmethylated GATC

                 Recruits MutU (helicase) and exonucleases

                 DNA pol III fills in the gap
                           Mismatch Repair in Human Cells

                                                           MSH2 and MSH6 bind to mismatch-
                                                           containing DNA and distinguish between
                                                           the template and newly synthesized strand

                                                           MMR complex identifies newly synthesized
                                                           strand by the presence of a 3’-terminus

                                                           MutLa introduces random nicks
                                                           at distal sites on the same strand

                                                           EXO1 at 5’-side of the mismatch activates
                                                           a 5’-3’ exonuclease and removes mismatch

                                                           The gap is filled in by DNA polymerase
                                                           and DNA ligase

                                                           Defective mismatch repair is the primary
                                                           cause of certain types of human cancers
from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-37
         Causes of and Responses to ds Breaks

                                              DSBs result from exogenous insults
                                              or normal cellular processes

                                              DSBs result in cell cycle
                                              arrest, cell death, or repair

                                              Repair of DSBs is by
                                              homologous recombination
                                              or nonhomologous end joining

from van Gent et al., Nature Rev.Genet. 2, 196 (2001)
       ATM Mediates the Cell’s Response to DSBs

                                                        DSBs activate ATM

                                                        ATM phosphorylation of p53,
                                                        NBS1 and H2AX influence cell
                                                        cycle progression and DNA repatr

from van Gent et al., Nature Rev.Genet. 2, 196 (2001)
Repair of ds Breaks by Homologous Recombination

                         ssDNAs with 3’ends are formed and
                         coated with Rad51, the RecA homolog

                         Rad51-coated ssDNA invades the
                         homologous dsDNA in the sister chromatid

                         The 3’-end is elongated by DNA polymerase,
                         and base pairs with ss 3-end of the other broken DNA

                         DNA polymerase and DNA ligase fills in gaps

                 from Lodish et al., Molecular Cell Biology, 5th ed. Fig 23-31
Repair of ds Breaks by Nonhomologous End Joining

                                                KU heterodimer recognizes
                                                DSBs and recruits DNA-PK

                                                Mre11 complex tethers ends
                                                together and processes DNA ends

                                                DNA ligase IV and
                                                XRCC4 ligates DNA ends

 from van Gent et al., Nature Rev.Genet. 2, 196 (2001)
        Structure of Rad50 and the Mre11 Complex

                                                       Mre11 complex maintains
                                                       genome integrity and
                                                       participates in HR and NHEJ

                                                       AT-like syndrome and Nijmegen
                                                       breakage syndrome are caused by
                                                       mutation in MRE11 or NBS1

                                                       Dimerization domain of Rad50 keeps
                                                       DNA fragments within close proximity

from de Jager and Kanaar, Genes Dev. 16, 2173 (2002)

X-ray-repair-cross-complementing defective repair in Chinese hamster mutant 4
          The SCID Mouse is Defective in DSB Repair

The SCID mouse carries a mutation preventing
the production of mature B and T cells

The phenotype is a defect in the development of the
immune system and hypersensitivity to ionizing radiation

The phenotype is caused by a mutation in DNA-PK
        Translesion Replication by DNA Polymerase V

                                         Translesion DNA synthesis occurs
                                         in the absence of Pol III

                                         Translesion DNA polymerases are
                                         error prone and exhibit weak processivity

                                         Most of the mutations caused by DNA
                                         damaging agents are caused by TLR

                                         TLR protects the genome from gross rearrangements

                                         Pol V is regulated by LexA and the SOS response

from Livneh, J.Biol.Chem. 276, 25639 (2001)
Expandable Repeats Form Unusual DNA Structures

                                              One of the complementary strands is
                                              more structure-prone than the other

                                              Unusual structural features
                                              predispose them to instability

                                              Stalling of lagging strand
                                              synthesis disrupts coordination
                                              with leading strand synthesis

        from Mirkin, Nature 447, 932 (2007)

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