DNA repair and mutagenesis

BIOL122a

Prof. Sue Lovett

Sources of mutation

• Natural polymerase error• Endogenous DNA damage

oxidative damage depurination

• Exogenous DNA damageradiationchemical 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

QuickTime™ and aGIF decompressorare needed to see this picture.

•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 Genotype Rifr mutants per 108 cells

Wild-type mut+ 5-10

mutD (dnaQ)

Pol III proofreading

4000-5000

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

NH2

O

O

H2C

O

ON

HN

O

O

O

H2C

O

O

deoxycytosine deoxyuracil

1’

2’3’

4’

5’

12

34

5

6

CH3

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 10-1000x• 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 MMRCH3 CH3

5'3' 5'

3'

Initiation

CH3 CH35'3' 5'

3'CH3 CH3

5'3' 5'

3'

MutS MutL MutH MutS MutL MutH

Excision

CH3 CH35'3' 5'

3'CH3 CH3

5'3' 5'

3'

UvrD + RecJ or ExoVIIUvrD + ExoI or ExoX or ExoVII

ResynthesisCH3 CH3

5'

3' 5'

3'CH3 CH3

5'

3' 5'

3'

PolIII + ligase PolIII + ligase

Mechanism of MMRCH3 CH3

5'3' 5'

3'

Initiation

CH3 CH35'3' 5'

3'CH3 CH3

5'3' 5'

3'

MutS MutL MutH MutS MutL MutH

Excision

CH3 CH35'3' 5'

3'CH3 CH3

5'3' 5'

3'

UvrD + RecJ or ExoVIIUvrD + ExoI or ExoX or ExoVII

ResynthesisCH3 CH3

5'

3' 5'

3'CH3 CH3

5'

3' 5'

3'

PolIII + ligase 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

CG

5’5’

5’

5’

5’

5’

5’

5’

Heat denature

A

T

G

C

5’5’

5’

5’

A5’

C5’

T5’

G5’

Cool renature

homoduplexes +

heteroduplexes

A

T

G

C

5’5’

5’

5’

Heat denature

CsCl gradients

T5’

G5’

“heavy strand”

“light strand”

Single heteroduplex

In bacteriophage lambda (40 kb):

Transfect, repair

G

C

A

T5’

5’

5’

5’

A

T

G

C

5’5’

5’

5’

Heat denature

CsCl gradients

T5’

G5’

“heavy strand”

“light strand”

hemi-methylated heteroduplex

Grow in Dam+: Grow in Dam-:

* * ** * *

* * * Transfect, Methyl-directed repair

5’

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 protein Human protein Probable function Rad14 XPA Binds damaged DNA after XPC or RNA pol II Rpa1,2,3   RPAp70,p32,p14  Stabilizes open complex (with Rad14/XPA); positions

nucleasesRad4  XPC  Works with hHR23B; binds damaged DNA;

recruits other NER proteinsRad23  hHR23B  Cooperates with XPC (see above); contains ubiquitin

domain; interacts with proteasome and XPC Ssl2 (Rad25) XPB 3' to 5' helicase Tfb1 p62 ? Tfb2 p52 ?  Ssl1  p44 DNA binding?Tfb4  p34  DNA binding?   Rad3   XPD  5' to 3' helicase Tfb3/Rig2 MAT1  CDK assembly factor Kin28  Cdk7 CDK; C-terminal domain kinase; CAK Ccl1  CycH Cyclin Rad2 XPG Endonuclease (3' incision); stabilizes full open complex Rad1 XPF Part of endonuclease (5' incision) Rad10 ERCC1 Part of endonuclease (5' incision)

Human NER

Rad1/10 Rad2 in 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 100-10,000 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. 2002 DNA mismatch repair and mutation avoidance

pathways. J. Cell. Physiol. 191: 28-41• *Harfe BD, Jinks-Robertson S. 2000 DNA mismatch repair and genetic instability.

Annu. Rev. Genet. 34: 359-399.• *Krokan, HE, Standal, R, Slupphaug, G. 1997 DNA glycosylases in the base excision

repair of DNA Biochem. J. 325: 1-16. • *De Laat, WL, Jaspers, NGJ, Hoeijmakers, JHJ. 1999 Molecular mechanism of

nucleotide excision repair. Genes Dev. 13: 768-785• Petit, C, Sancar, A. 1999 Nucleotide excision repair: from E. coli to man. Biochimie 81:

15-25• *Goodman, MF, Tippin, B. 2000. Sloppier copier DNA polymerases involved in

genome repair. Curr. Opin. Genet. Dev. 10:162-168.• *Friedberg, EC, Wagner, R, Radman, M. Specialized DNA polymerases, cellular

survival and the genesis of mutations. Science 296: 1627-1630. • Goodman, MF 2002. Error-prone repair DNA polymerases in prokaryotes and

eukaryotes. Annu. Rev. Biochem. 71: 17-50