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DNA repair and mutagenesis

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Title: DNA repair and mutagenesis


1
DNA repair and mutagenesis
  • BIOL122a
  • Prof. Sue Lovett

2
Sources of mutation
  • Natural polymerase error
  • Endogenous DNA damage oxidative damage
    depurination
  • Exogenous DNA damage radiation chemical adducts
  • Error-prone DNA repair

3
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.

4
Common features of DNA polymerases
  • Right hand palm, fingers, thumb
  • Palm --gt phoshoryl transfer
  • Fingers --gt template and incoming nucleoside
    triphosphate
  • Thumb --gt DNA positioning, processivity and
    translocation
  • Some polymerase have associated 3 to 5
    exonuclease proofreading activity in a second
    domain

5
Structures of 4 polymerase classes
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  • Fidelity is increased by action of 3 to 5
    exonuclease proofreading activity
  • Active site of exo is 30 Å from pol, below palm

8
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
9
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

10
Mechanism of BER
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NH2
O
4
3
HN
N
5
O
O
2
1
6
O
O
N
N
H2C
H2C
5
O
O
glycosidic bond
1
4
3
2
O
O
deoxycytosine
deoxyuracil
13
Types of lesions repaired by BER
  • Oxidative lesions 8-oxo-G, highly mutagenic,
    mispairs with A, producing GC --gt TA
    transversions example MutY, MutMFpg from E. coli
  • Deoxyuracil from misincorporation of dU or
    deamination of dC--gtdU, 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

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Flipping out mechanism
16
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

17
Mechanism of MMR
18
Mechanism of MMR
19
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 CC and
    short frameshift loops lt4 bp
  • Transition mispairs GT and AC and one base
    loops are particularly well-recognized (these are
    also the most common polymerase errors)

20
Structure of MutS bound to DNA
60 kink in DNA Widens minor groove, narrows
major groove
21
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
    GATC 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

22
A
G
5
5
5
5
T
C
Heat denature
A
5
G
5
5
C
5
T
Cool renature
A
G
5
5
5
5
T
C
homoduplexes
A
G
5
5
5
5
C
T
heteroduplexes
23
In bacteriophage lambda (40 kb)
A
G
5
5
5
5
T
C
Heat denature
light strand
CsCl gradients
heavy strand
Single heteroduplex
G
5
5
T
24
Grow in Dam
Grow in Dam-
A
G

5
5
5
5

T
C
Heat denature
light strand
CsCl gradients
heavy strand
hemi-methylated heteroduplex
G
5
5

T
25
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

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34
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

35
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)

36
Recognition and binding
UvrA acts as classical molecular matchmaker
37
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
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) XPB 3' to 5' helicase  Tfb1 p62 ?  Tfb2
p52 ?   Ssl1  p44 DNA binding? Tfb4  p34  D
NA 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 Endon
uclease (3' incision) stabilizes full open
complex  Rad1 XPF Part of endonuclease (5'
incision)  Rad10 ERCC1 Part of endonuclease (5'
incision)
38
Human NER
Rad1/10
Rad2 in S. cerevisiae
39
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 UmuDC (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

40
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

41
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42
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 ?
43
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. 10162-168.
  • 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
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