Replication

Biochemistry 3107 - Fall 2004

DNA Repair Repair

Recombination

How do mutations arise?

Misincorporation during DNA synthesis

As a result of chemical or environmental damage

BRCA2 Function in DNA Insertions by mobile elements

Binding and Recombination
from a BRCA2-DSS1-ssDNA
Structure
How are mutations repaired?
Haijuan Yang, Philip D. Jeffrey, Julie
Miller, Elspeth Kinnucan, Yutong Sun,
Nicolas H. Thomä, Ning Zheng, Phang-
Lang Chen, Wen-Hwa Lee, and Nikola P.
Pavletich
Repair during and immediately after DNA synthesis
Science Sep 13 2002: 1837-1848.
Direct reversal or by excising the mutation

Types of Mutations
How is the alteration detected?
Simplest are changes to single bases
How is it properly repaired?

• How is repair accomplished quickly? TRANSITIONS

• How does the cell distinguish parental and

daughter strands when fixing replication
errors?
• How does the cell fix large breaks when the
original sequence has been lost?
• How does the cell cope with lesions that block
TRANSVERSIONS
replication?

MBG-9-1

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Types of Mutations Types of Mutations
More complex are Insertion/Deletion Mutations More complex are Insertion/Deletion Mutations

• occur within reading frames

• deletions can be very large.
• essential genes can’t be deleted.
INSERTIONS & DELETIONS
• deletions arise as a result of
recombination reactions.
• insertions caused by Insertion
Sequences or Transposons
• insertions will usually inactivate a
gene.
• involve the insertion or
deletion of one or a few base
pairs.
FRAMESHIFT MUTATIONS
• occur as a result of "slippage"
during DNA replication.
• arise as a result of
intercalating mutagens such
as ethidium bromide and
acridine orange.

Fidelity of DNA Replication

DNA microsatellite sequences are prone to mutation
• DNA replication is remarkably accurate
Expansion of triplet repeats • Errors occur only once every 109-1010 nucleotides
incorporated
CGG
CAG • DNA polymerases are not so accurate
• Errors occur once every 104-105 nucleotides incorporated

• Muscular dystrophy
• The proofreading activity of a polymerase will improve the
• Fragile X syndrome overall error rate by 102-103
• Huntington’s disease (CAG) • this still leaves a difference of 102-103 between the error rates
of DNA synthesis and replication

Mutations are made permanent by later rounds of replication DNA mismatch repair systems
must be able to distinguish newly-
synthesized DNA from parental
DNA

newly-synthesized DNA strands

are non-methylated while
parental DNA strands are
methylated

newly-synthesized DNA is
• mismatch repair systems fix the errors made during hemimethylated.
replication but not caught by proofreading.

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 DNA mismatch repair systems
How and where is DNA must be able to scan the genome
and detect mismatches
methylated?

dam methylase MutS recognises mismatches by

detecting distortion in DNA
GATC

dam- mutants of E. coli have

increased rates of
spontaneous mutation DNA Mismatch Repair

MBG-9-5

MutS recognises mismatches by MutS recognises mismatches by

detecting distortion in DNA detecting distortion in DNA

The DNA distortion produces a

conformational change in MutS

The complex attracts MutL

MutL activates MutH which nicks

DNA

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• MutH is structurally
related to restriction
endonucleases

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 MBG-9-6

• Exonuclease I (3’-> 5’) or Exonuclease VII (5’-> 3’) removes DNA

between the the nick caused by MutH and the site of the mismatch.
• UvrD helicase unwinds DNA

• The gap is repaired by DNA polymerase III and sealed by DNA

ligase

The Cancer Connection

Eukaryotes
Human nonpolyposis
colorectal cancer is
associated with defects in a MutS & MutL homologues - HNPCC
human counterpart to MutS
(HNPCC Type 1) or MutL No dam methylase in eukaryotes or many bacteria
(HNPCC Type 2)

Recruited to sliding clamp (PCNA)

DNA Mismatch Repair
Genetic polymorphisms
linked to HNPCC (type 1) Repair of mismatched Okazaki fragments
were first identified in a
Newfoundland family and in a
New Zealand family

Repair Systems
How do mutations arise?

Direct Reversal Misincorporation during DNA synthesis

As a result of chemical or environmental damage
Excision of the modified base Insertions by mobile elements

Excision of modified nucleotides - Nucleotide Excision Repair

Excision of (un)modified nucleotides - DNA Mismatch Repair

Repair by recombination - Postreplication Repair

SOS Repair

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DNA is damaged by the
action of water

deamination

MBG-9-7 VVP24-25

DNA is damaged by the DNA is damaged by the

action of water action of water

deamination deamination

Depurination
formation of abasic sites

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DNA is damaged by Methylation & ethylation UV radiation causes the

alkylation, oxidation and also on phosphates formation of thymine dimers
radiation

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Base analogs and Base analogs and
Intercalating agents cause Intercalating agents cause
mutations mutations

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Base analogs How do mutations arise?

and
Intercalating
agents cause Misincorporation during DNA synthesis
mutations
As a result of chemical or environmental damage
Insertions by mobile elements

How are mutations repaired?

Repair during and immediately after DNA synthesis

Direct reversal or by excising the mutated base

Table 9-1

DIRECT REVERSAL
Mismatch repair Replication errors
Photoreactivation

Photoreactivation Pyrimidine dimers

Base excision repair Damaged base

Nucleotide excision repair Pyrimidine dimer

Bulky adduct on a base

Double-strand break repair Double-strand breaks

Translesion DNA synthesis Pyrimidine dimer or

apurinic site
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 DIRECT REVERSAL EXCISION REPAIR SYSTEMS
base excision repair
Removal of methyl groups
Removal of damaged bases by
glycosylases and AP endonucleases

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EXCISION REPAIR SYSTEMS EXCISION REPAIR SYSTEMS

base excision repair base excision repair

The damaged base Failsafe systems

is flipped out of the exist in some cases
double helix
MutY
MutM

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EXCISION REPAIR SYSTEMS EXCISION REPAIR SYSTEMS

nucleotide excision repair nucleotide excision repair

Recognise distortions in DNA

rather than specific changes
UvrA2B complex scans DNA
• Thymine dimer
When it encounters a distortion,
• Bulky chemical adduct UvrA exits

UvrB recruits UvrC which nicks

UvrA2B complex scans DNA 8 nt on the 5’ side and 4-5 nt on
the 3’ side
When it encounters a distortion,
UvrA exits UvrD helicase unwinds and
removes a 13 nt ssDNA
segment

DNA PolI and DNA ligase fill the

gap

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 EXCISION REPAIR SYSTEMS EXCISION REPAIR SYSTEMS
nucleotide excision repair nucleotide excision repair

Transcription-coupled repair
UvrA XPC Detects distortions
Excision repair proteins help
rescue stalled RNA polymerase
UvrB XPA Helicase activities to
XPD form bubble
RPD Transcription factor TFIIH
include XPA and XPD

UvrC ERCC1-XPF Cleavage on 5’ side

XPG Cleavage on 3’ side

Removal of 24-32 nt
piece of DNA

RECOMBINATION REPAIR SYSTEMS TRANSLESION DNA SYNTHESIS

postreplication repair “emergency” repair

Occurs after replication Replication machinery tries

to bypass sites of damage

Use the information in an

intact DNA molecule to fix a Catalyzed by special DNA
lesion (e.g.) in the other. polymerases

DNA pol IV
Similar to double-stranded
break repair DNA pol V = UmuD’2C

Highly error-prone

VVP24-30

TRANSLESION DNA SYNTHESIS TRANSLESION DNA SYNTHESIS

“emergency” repair “emergency” repair

Highly error-prone

Bases are
incorporated
independent of base
pairing

SOS response

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 Induced in response to major Reminder: PolV is an SOS induced enzyme
damage to the bacterial DNA
Also induced by agents which
inhibit DNA replication • Two SOS genes, umuC and
umuD, code for DNA
Polymerase V (UmuD'2C)
Complex system
> 20 genes
Two are important regulator
genes:
lexA r e c A

LexA is a repressor that blocks

the transcription of all of the
SOS Repair other SOS repair genes,
including recA and lexA itself