dna repair and mutagenesis biol122a prof. sue lovett
TRANSCRIPT
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