Download Repair mechanisms - Pennsylvania State University

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
HN
O
N
6
H
5 CH3
HN
O
N
6
H
Thymine
Thymine
d-ribose
d-ribose
Photolyase
breaks the
bonds
between the
dTs
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
100wt
50u vr -
uvr -
% S u r v ivo r s
10D 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'
3'
+
A
A
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'
ATP
5'
3'
UvrD (helicase II) unwinds and liberates the damaged fragment
+
B C
Fill in with polymerase and ligate
5'
3'
dNTPs
DNA polymerase I fills in the gap
5'
3'
NAD
5'
3'
DNA ligase
Mutations in excision repair in eukaryotes
can cause xeroderma pigmentosum (XP)
Human
Gene
XPA
XPB
XPC
XPD
XPE
XPF
XPG
Protein Function
Binds damaged DNA
Helicase, Component of TFIIH
DNA damage sensor
Helicase, Component of TFIIH
Binds damaged DNA
Works with ERRCI to cut DNA
Cuts DNA
Analogous
to E. coli:
UvrA/UvrB
UvrD
UvrD
UvrA/UvrB
UvrB/UvrC
UvrB/UvrC
2b. Base excision repair
Incorrect or
damaged base
P
P
A
T
P
P
G
C
P
T
A
P
P
U
A
P
P
C
G
P
P
A
T
P
P
T
A
P
P
P
A
T
P
G
C
P
P
C
G
P
P
P
G
C
P
P
G
C
P
P
T
A
P
A
T
P
P
P
A
T
P
P
C
G
P
P
G
C
P
P
T
A
P
+
A
T
P
U
P
AP endonuclease cuts the phosphodiester backbone 5' to the
AP site.
P
P
OH
P
C
G
A
P
A
T
P
P
C
G
A
T
A
P
P
P
primer
P
Glycosylase recognizes damaged base and cuts the bond to the
sugar in the DNA backbone.
P
G
C
G
C
P
AP site
P
P
P
P
G
C
P
P
A
T
P
P
C
G
P
P
G
C
P
P
T
A
P
A
T
P
P
Excision and filling in by DNA PolI
primer
P
P
A
T
P
G
C
P
P
P
OH
T
A
P
C
G
A
P
P
P
P
G
C
P
P
A
T
P
P
C
G
P
P
G
C
P
P
T
A
P
A
T
P
P
DNA pol ymerase I removes the damaged strand (5' to 3'
exonuclease) and fills in correct sequence (polymerase).
P
P
A
T
P
G
C
P
P
T
A
P
P
T
A
P
P
C
G
P
P
A
T
P
G
C
P
P
T
A
P
P
A
T
P
P
P
C
G
P
P
OH
G
C
P
nick
T
A
P
A
T
P
P
DNA ligase seals the nick
P
P
C
G
T
A
P
G
C
P
NAD
P
P
P
P
G
C
P
P
A
T
P
P
C
G
P
P
G
C
P
P
T
A
P
Repaired DNA
A
T
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. GT 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'
5'
3'
Replication past a damaged site leaves a
gap on the opposite strand plus one
correct copy.
Gap is repaired by retrieving DNA from the correct copy of the chromosome,
using DNA recombination.
5'
3'
5'
3'
Damaged site can be
repaired by excision repair
(e.g. UvrABC)
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
damage
DNA
replication
DNA Pol III
UmuD 2
UmuC
beta
UV damage, increase
RecA:ssDNA
Activate protease
Induce umuC+, umuD+
epsilon
DNA damage checkpoint control
Graham Walker
Translesional
synthesis
(error-prone)
UmuD’2
UmuC
Pol III alpha
Polymerase for
translesional synthesis
SOS response is controlled by LexA and
SOS response is controlled by LexA and RecA
RecA
OFF
LexA
LexA
recA
Repressed
LexA
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
+
RecA
RecA
RecA
LexA
e.g. RecA, UvrA, UvrB, UmuC
active proteins
de-repressed
recA
lexA
target gene
LexA, RecA in the SOS response