DNA repair and mutagenesis BIOL122a Prof. Sue Lovett

Sources of mutation Natural polymerase error Endogenous DNA damage oxidative damage depurination Exogenous DNA damage radiation chemical adducts “Error-prone” DNA repair

Cellular protection from DNA damage Natural errors: polymerase base selection, proofreading, mismatch repair Endogenous/exogenous DNA damage: base excision repair, nucleotide excision repair, (recombination, polymerase bypass) Recombination and polymerase bypass do not remove damage but remove its block to replication. Polymerase bypass is itself often mutagenic.

Common features of DNA polymerases Right hand: “palm”, “fingers”, “thumb” Palm --> phoshoryl transfer Fingers --> template and incoming nucleoside triphosphate Thumb --> DNA positioning, processivity and translocation Some polymerase have associated 3’ to 5’ exonuclease “proofreading” activity in a second domain

Structures of 4 polymerase classes

Fidelity is increased by action of 3’ to 5’ exonuclease “proofreading” activity Active site of exo is 30 Å from pol, below palm

Contribution of proofreading, base excision repair and MMR to mutation avoidance GenotypeRif r mutants per 10 8 cells Wild-type mut mutD (dnaQ) Pol III proofreading mutS MMR 760 mutY mutM 8-oxoG BER 8200

Base excision repair (BER) Major pathway for repair of modified bases, uracil misincorporation, oxidative damage Various DNA glycosylases recognize lesion and remove base at glycosidic bond, thereby producing an “abasic” or AP (apurinic/ apyrimidinic) site by base “flipping out” One of several AP endonucleases incises phosphodiesterase backbone adjacent to AP site AP nucleotide removed by exonuclease/dRPase and patch refilled by DNA synthesis and ligation

Mechanism of BER

N N NH 2 O O H2CH2C O O N HN O O O H2CH2C O O deoxycytosine deoxyuracil 1’ 2’ 3’ 4’ 5’ CH 3 thymine glycosidic bond

Types of lesions repaired by BER Oxidative lesions; 8-oxo-G, highly mutagenic, mispairs with A, producing GC --> TA transversions example MutY, MutM=Fpg from E. coli Deoxyuracil: from misincorporation of dU or deamination of dC-->dU, example Ung, uracil N- glycosylase Various alkylation products e. g. 3-meA These lesions are not distorting and do not block DNA polymerases Spontaneous depurination (esp. G) yield abasic sites that are repaired by second half of BER pathway

“Flipping out” mechanism

Mismatch repair (MMR) Despite extraordinary fidelity of DNA synthesis, errors do persist Such errors can be detected and repaired by the post- replication mismatch repair system Prokaryotes and eukaryotes use a similar mechanism with common structural features Defects in MMR elevate spontaneous mutation rates x Defects in MMR underlie human predisposition to colon and other cancers (“HNPCC”) MMR also processes mispairs that result from heteroduplex DNA formed during genetic recombination: act to exclude “homeologous” recombination

Mechanism of MMR CH 3 3 5' 3'5' 3' Initiation CH 3 3 5' 3'5' 3' CH 3 3 5' 3'5' 3' MutS MutL MutH Excision CH 3 3 5' 3'5' 3' CH 3 3 5' 3'5' 3' UvrD + RecJ or ExoVIIUvrD + ExoI or ExoX or ExoVII Resynthesis CH 3 3 5' 3'5' 3' CH 3 3 5' 3'5' 3' PolIII + ligase

Mechanism of MMR CH 3 3 5' 3'5' 3' Initiation CH 3 3 5' 3'5' 3' CH 3 3 5' 3'5' 3' MutS MutL MutH Excision CH 3 3 5' 3'5' 3' CH 3 3 5' 3'5' 3' UvrD + RecJ or ExoVIIUvrD + ExoI or ExoX or ExoVII Resynthesis CH 3 3 5' 3'5' 3' CH 3 3 5' 3'5' 3' PolIII + ligase

Basis of MMR recognition MutS dimer (in yeast, Msh2/Msh3 or Msh2/Msh6 heterodimer) By DNA binding expts in vitro and DNA heteroduplex repair expts in vivo: MMR can recognize all base substitutions except C:C and short frameshift loops <4 bp Transition mispairs G:T and A:C and one base loops are particularly well-recognized (these are also the most common polymerase errors)

Structure of MutS bound to DNA 60° kink in DNA Widens minor groove, narrows major groove

The problem of strand discrimination MMR can only aid replication fidelity if repair is targeted to newly synthesized strand In E. coli, this is accomplished by the transient lack of methylation of adenines in GA*TC motifs (by the “Dam” methylase) MutH endonuclease cleaves only unmethylated GATC sites, allowing entry on newly synthesized strand dam mutants are “mutators” and show random repair of either DNA strand In other bacteria and in eukaryotes, the basis of strand discrimination is not understood, although entry at nicks in discontinuously synthesized DNA has been proposed

A T G C A T C G 5’ Heat denature A T G C 5’ A C T G Cool renature homoduplexes + heteroduplexes

A T G C 5’ Heat denature CsCl gradients T 5’ G “heavy strand” “light strand” Single heteroduplex In bacteriophage lambda (40 kb): Transfect, repair G C A T 5’

A T G C Heat denature CsCl gradients T 5’ G “heavy strand” “light strand” hemi-methylated heteroduplex Grow in Dam + :Grow in Dam - : * * * Transfect, Methyl-directed repair 5’ * * * A T

Various Msh and Mlh (Pms1) heterodimers vs. MutS and MutL homodimers Msh2/6 specialized for base substitution mispairs; Msh2/3 for loop mispairs No MutH, Dam; basis for strand discrimination unknown Basis of excision (comparable to UvrD and Exos) incompletely understood Comparison of eukaryotic vs. prokaryotic MMR

Nucleotide excision repair (NER) Recognizes bulky lesions that block DNA replication (i. e. lesions produced by carcinogens)- -example, UV pyrimidine photodimers Common distortion in helix Incision on both sides of lesion Short patch of DNA excised, repaired by repolymerization and ligation In E. coli, mediated by UvrABCD Many more proteins involved in eukaryotes Can be coupled to transcription (TCR, “transcription coupled repair”) Defects in NER underlie Xeroderma pigmentosum

Xeroderma pigmentosum Autosomal recessive mutations in several complementation groups Extreme sensitivity to sunlight Predisposition to skin cancer (mean age of skin cancer = 8 yrs vs. 60 for normal population)

Recognition and binding UvrA acts as classical “molecular matchmaker” Incision Nicks delivered 3’ and 5’ to lesion by UvrBC Excision and repair Short fragment released by helicase action

Proteins Required for Eukaryotic Nucleotide Excision Repair S. cerevisiae proteinHuman proteinProbable function Rad14XPABinds damaged DNA after XPC or RNA pol II Rpa1,2,3 RPAp70,p32,p14 Stabilizes open complex (with Rad14/XPA); positions nucleases Rad4 XPC Works with hHR23B; binds damaged DNA; recruits other NER proteins Rad23 hHR23B Cooperates with XPC (see above); contains ubiquitin domain; interacts with proteasome and XPC Ssl2 (Rad25)XPB3' to 5' helicase Tfb1p62? Tfb2p52? Ssl1 p44DNA binding? Tfb4 p34 DNA binding? Rad3 XPD 5' to 3' helicase Tfb3/Rig2MAT1 CDK assembly factor Kin28 Cdk7CDK; C-terminal domain kinase; CAK Ccl1 CycHCyclin Rad2XPGEndonuclease (3' incision); stabilizes full open complex Rad1XPFPart of endonuclease (5' incision) Rad10ERCC1Part of endonuclease (5' incision)

Human NER Rad1/10 Rad2in S. cerevisiae

Lesion bypass polymerization Replication-blocking lesions such as UV photodimers can be repaired by NER but pose a serious problem if they are in ssDNA As a last resort, cells employ “bypass” polymerases with loosened specificity In E. coli: DinB (PolIV) and UmuD’C (Pol V); homologs in eukaryotes; mutated in XPV These polymerases are “error-prone” and are responsible for UV-induced mutation Expression and function highly regulated: dependent on DNA damage

Characteristics of lesion bypass polymerases Error rate ,000 x higher on undamaged templates Lack 3’ to 5’ proofreading exonuclease activity Exhibit distributive rather than processive polymerization (nt. incorporated per binding event) Support translesion DNA synthesis in vitro

Table 1. Low-fidelity copying of undamaged DNA by specialized DNA polymerases from human cells. [Adapted from P. J. Gearhart and R. D. Wood, Nature Rev. Immunol. 1, 187 (2001)] DNA polymerase Gene Infidelity on undamaged DNA templates (relative to pol  = ~1)  POLB ~50  REV3L ~70  POLK ~580  POLH ~2,000  POLI ~20,000 POLL ? µ POLM ?  POLQ ? Rev1 REV1L ?

Further references Friedberg. DNA repair and mutagenesis. ASM Press, Washington, D. C. *Marti TM, Kunz, C, Fleck O DNA mismatch repair and mutation avoidance pathways. J. Cell. Physiol. 191: *Harfe BD, Jinks-Robertson S DNA mismatch repair and genetic instability. Annu. Rev. Genet. 34: *Krokan, HE, Standal, R, Slupphaug, G DNA glycosylases in the base excision repair of DNA Biochem. J. 325: *De Laat, WL, Jaspers, NGJ, Hoeijmakers, JHJ Molecular mechanism of nucleotide excision repair. Genes Dev. 13: Petit, C, Sancar, A Nucleotide excision repair: from E. coli to man. Biochimie 81: *Goodman, MF, Tippin, B Sloppier copier DNA polymerases involved in genome repair. Curr. Opin. Genet. Dev. 10: *Friedberg, EC, Wagner, R, Radman, M. Specialized DNA polymerases, cellular survival and the genesis of mutations. Science 296: Goodman, MF Error-prone repair DNA polymerases in prokaryotes and eukaryotes. Annu. Rev. Biochem. 71: 17-50