Transcript PPT DNA repair – Life Sciences at Brandeis University

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
QuickT ime™ and a GIF decompressor are 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
Wild-type mut+
Rifr mutants per 108 cells
5-10
mutD (dnaQ)
Pol III proofreading
mutS
MMR
mutY mutM
8-oxoG BER
4000-5000
760
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
NH2
4
3
5’
O
H2 C
O
2
1
N
4’
6
CH3
HN
5
N
O
O
O
O
H2 C
O
N
1’
3’
glycosidic bond
2’
O
deoxycytosine
O
deoxyuracil
thymine
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 Nglycosylase
• 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 postreplication mismatch repair system
• Prokaryotes and eukaryotes use a similar mechanism with
common structural features
• Defects in MMR elevate spontaneous mutation rates 101000x
• 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
5'
3'
MutS MutL MutH
5'
3'
CH3
5'
3'
CH3
3'
5'
5'
3'
5'
3'
Excision
CH3
3'
5'
PolIII + ligase
CH3
3'
5'
MutS MutL MutH
Initiation
UvrD + ExoI or ExoX or ExoVII
CH3
CH3
CH3
5'
3'
3'
5'
5'
3'
3'
5'
UvrD + RecJ or ExoVII
CH3
CH3
3'
5'
PolIII + ligase
Resynthesis
CH3
CH3
CH3
CH3
CH3
3'
5'
Mechanism of MMR
5'
3'
MutS MutL MutH
5'
3'
CH3
5'
3'
CH3
3'
5'
5'
3'
5'
3'
Excision
CH3
3'
5'
PolIII + ligase
CH3
3'
5'
MutS MutL MutH
Initiation
UvrD + ExoI or ExoX or ExoVII
CH3
CH3
CH3
5'
3'
3'
5'
5'
3'
3'
5'
UvrD + RecJ or ExoVII
CH3
CH3
3'
5'
PolIII + ligase
Resynthesis
CH3
CH3
CH3
CH3
CH3
3'
5'
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
5’
5’
T
Heat denature
5’
5’
5’
A
C
C
5’
C
5’
Cool
renature
A
T
5’
A
G
T
5’
G
5’
G
5’
5’
homoduplexes
+
5’
5’
heteroduplexes
5’
C
G
T
5’
5’
In bacteriophage lambda (40 kb):
A
G
5’
5’
T
Heat denature
5’
C
“light strand”
CsCl gradients
“heavy strand”
Single heteroduplex
G
5’
5’
T
5’
G
5’
C
Transfect,
repair
5’
A
5’
T
Grow in Dam+:
5’
A
*
* T
*
*
Grow in Dam-:
*
*
5’
5’
Heat denature
G
5’
C
“light strand”
CsCl gradients
“heavy strand”
hemi-methylated heteroduplex
5’
G
* T
5’
* *
5’
Transfect,
Methyl-directed repair
A
*T
* *
5’
Comparison of eukaryotic vs.
prokaryotic MMR
• 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
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
Rad14
XPA
Rpa1,2,3
RPAp70,p32,p14
Rad4
XPC
Rad23
hHR23B
Ssl2 (Rad25)
Tfb1
Tfb2
Ssl1
Tfb4
Rad3
Tfb3/Rig2
Kin28
Ccl1
Rad2
Rad1
Rad10
XPB
p62
p52
p44
p34
XPD
MAT1
Cdk7
CycH
XPG
XPF
ERCC1
Probable function
Binds damaged DNA after XPC or RNA pol II
Stabilizes open complex (with Rad14/XPA); positions
nucleases
Works with hHR23B; binds damaged DNA;
recruits other NER proteins
Cooperates with XPC (see above); contains ubiquitin
domain; interacts with proteasome and XPC
3' to 5' helicase
?
?
DNA binding?
DNA binding?
5' to 3' helicase
CDK assembly factor
CDK; C-terminal domain kinase; CAK
Cyclin
Endonuclease (3' incision); stabilizes full open complex
Part of endonuclease (5' incision)
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 e = ~1)
-----------------------------------------------------------------------b
POLB
~50
z
REV3L
~70
k
POLK
~580
h
POLH
~2,000
i
POLI
~20,000
l
POLL
?
µ
POLM
?
q
POLQ
?
Rev1
REV1L
?
Further references
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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