Repair mechanisms

1. Reversal of damage
2. Excision repair
3. Mismatch repair
4. Recombination repair
5. Error-prone repair
6. Restriction-modification systems
 1. Reversal of damage
• Enzymatically un-do the damage
• a) Photoreactivation
• b) Removal of methyl groups
Photolyase breaks apart pyrimidine dimers

O O
5 CH3 5 CH3
HN HN

6 6 Photolyase
O N O N
breaks the
H H bonds
Thymine Thymine between the
dTs
d-ribose d-ribose
 1a. Photoreactivation
• Photolyase: binds a pyrimidine dimers and
catalyzes a photochemical reaction
• Breaks the cyclobutane ring and reforms
two adjacent T’s
• 2 subunits, encoded by phrA and phrB.
 2. Excision repair
• General Process:
– remove damage (base or DNA backbone)
– ss nick/gap provides 3’OH for DNA Pol I
initiation
– DNA ligase seals nick
• Nucleotide excision repair:
– Cut out a segment of DNA around a damaged
base.
• Base excision repair:
– Cut out the base, then cut next to the
apurinic/apyrimidinic site, and let DNA Pol I
repair
Discovery of mutants defective in DNA
repair

100-

wt
50-
u vr -
% S u r v ivo r s uvr -

10-

D o se o f U V
 polA mutants are defective in repair

wt

polA mutant

DNA synthesis in vitro Survival after UV in vivo

 UvrABC excision repair

damaged site
5'
3'
(UvrA) 2UvrB recognizes the damaged site

A A
B
5'
3'
ATP (UvrA) dissociates
2
B
5' + A A
3'
 Cleavage and helicase
B
5'
3'
UvrC binds UvrB at the damaged site

B C
5'
3'
ATP UvrBC nicks both 5' and 3' to the damaged site

B C
5'
3'
UvrD (helicase II) unwinds and liberates the damaged fragment
ATP

+ B C
5'
3'
 Fill in with polymerase and ligate

5'
3'
dNTPs DNA polymerase I fills in the gap

5'
3'
NAD DNA ligase

5'
3'
 Mutations in excision repair in eukaryotes
can cause xeroderma pigmentosum (XP)

Human Analogous
Gene Protein Function to E. coli:
XPA Binds damaged DNA UvrA/UvrB
XPB Helicase, Component of TFIIH UvrD
XPC DNA damage sensor
XPD Helicase, Component of TFIIH UvrD
XPE Binds damaged DNA UvrA/UvrB
XPF Works with ERRCI to cut DNA UvrB/UvrC
XPG Cuts DNA UvrB/UvrC
 2b. Base excision repair
Incorrect or damaged base

P P P P P P P P P P P

A G T U C G A C G T A
T C A A G C T G C A T
P P P P P P P P P P P

Glycosylase recognizes damaged base and cuts the bond to the

AP site sugar in the DNA backbone.

P P P P P P P P P P P

A G T C G A C G T A + U
T C A A G C T G C A T
P P P P P P P P P P P

AP endonuclease cuts the phosphodiester backbone 5' to the

primer AP site.
P

P P P OH P P P P P P P

A G T C G A C G T A
T C A A G C T G C A T
P P P P P P P P P P P
Excision and filling in by DNA PolI
primer P

P P P OH P P P P P P P

A G T C G A C G T A
T C A A G C T G C A T

P P P P P P P P P P P

DNA pol ymerase I removes the damaged strand (5' to 3'

exonuclease) and fills in correct sequence (polymerase).

P
P P P P P P P P P OH P
nick
A G T T C G A C G T A
T C A A G C T G C A T
P P P P P P P P P P P

NAD DNA ligase seals the nick

P P P P P P P P P P P

A G T C G A C G T A Repaired DNA
T
T C A A G C T G C A T
P P P P P P P P P P P
 3. Mismatch repair
• Action of DNA polymerase III (including
proofreading exonuclease) results in 1
misincorporation per 108 bases synthesized.
• Mismatch repair reduces this rate to 1
change in every 1010 or 1011 bases.
• Recognize mispaired bases in DNA, e.g. G-
T or A-C base pairs
• These do not cause large distortions in the helix:
the mismatch repair system apparently reads the
sequence of bases in the DNA.
 Role of methylation in discriminating
parental and progeny strands
• dam methylase acts on the A of GATC (note
that this sequence is symmetical or
pseudopalindromic).
• Methylation is delayed for several minutes
after replication.
• Mismatch repair works on the un-methylated
strand (which is newly replicated) so that
replication errors are removed preferentially.
 Action of MutS, MutL, MutH

• MutS: recognizes the mismatch

(heteroduplex)
• MutL: a dimer; in presence of ATP, binds to
MutS-heteroduplex complex to activate
MutH
• MutH: endonuclease that cleaves 5' to the
G in an unmethylated GATC, leaves a nick
MutH, L,
S action
in
mismatch
repair
#1
MutH, L, S action in mismatch repair #2
Mismatch repair: Excision of the
misincorporated nucleotide
 Eukaryotic homologs in mismatch
repair
• Human homologs to mutL (hMLH1) and
mutS (hMSH2, hMSH1) have been
discovered, because ...
• Mutations in them can cause one of the
most common hereditary cancers,
hereditary nonpolyposis colon cancer
(HNPCC).
 4. Recombination repair: retrieval of
information from a homologous chromosome
Recombination repair, a system for retrieval of information

damaged site
5'
3' Replication past a damaged site leaves a
gap on the opposite strand plus one
5' correct copy.
3'
Gap is repaired by retrieving DNA from the correct copy of the chromosome,
using DNA recombination.

5'
3' Damaged site can be
repaired by excision repair
5' (e.g. UvrABC)
3'
Gap in the correct copy is filled in by DNA polymerase.

5'
3'
 5. Error-prone repair
• Last resort for DNA repair, e.g when repair has
not occurred prior to replication. How does the
polymerase copy across a non-pairing, mutated
base, or an apyrimidinic/apurinic site?
– DNA polymerase III usually dissociates at a nick or a
lesion.
– But replication can occur past these lesions, especially
during the SOS reponse ("Save Our Ship").
• This translesion synthesis incorporates random
nucleotides, so they are almost always mutations
(3/4 times)
 Role of umuC and umuD genes in
error-prone repair
• Named for the UV nonmutable phenotype
of mutants with defects in these genes.
• Needed for bypass synthesis; mechanism
is under investigation. E.g. these proteins
may reduce the template requirement for
the polymerase.
• UmuD protein is proteolytically activated by
LexA.
 UmuC, UmuD in error-prone repair

UV DNA Translesional
damage replication synthesis
(error-prone)
DNA Pol III

UV damage, increase
UmuD 2 RecA:ssDNA UmuD’2
UmuC UmuC
Activate protease
Induce umuC+, umuD+

epsilon Pol III alpha

beta
DNA damage checkpoint control Polymerase for
translesional synthesis
Graham Walker
 SOS response is controlled by LexA and
RecA
SOS response is controlled by LexA and RecA

OFF LexA

LexA Repressed
LexA
recA lexA target gene

e.g. recA, lexA, uvrA, uvrB, umuC

RecA

ON RecA is activated in the presence of damaged DNA. It serves as a co-protease to activate a latent,
self-cleaving proteolytic activity in LexA, thereby removing the repressor from SOS inducible genes.

RecA
+ cleaved LexA e.g. RecA, UvrA, UvrB, UmuC

RecA RecA RecA LexA active proteins

de-repressed
recA lexA target gene
LexA, RecA in the SOS response