Chapter 20: Systems that safeguard DNA

1
Molecular GeneticsPCB4522 Spring 2007Lecture
9chapter 15Repair systemssection 15.20
2
Repair systems correct damage to DNA(Genes VIII
pp. 447-463)
Note Fig. references correspond to Genes VII.
Types of damage 1. single base changes do not
interfere with transcription or replication.
examples misincorporation or deamination create
mismatch. Mismatch persists only until next
replication, one copy is permanently mutated. 2.
structural distortions provide impediment to
replication or transcription. examples thymidine
dimers by UV, alkylation of G, single strand
nick, removal of a base. Since replication is
impaired, must be removed by some other mechanism
before next replication.

Fig. 15.33
Fig. 15.33
3

Fig. 15.33
Corrected by removing A or G
4

Fig. 15.34-36
5
Types of repair systems 1. Direct repair
Photoreactivation of pyrimidine dimers rare, but
is important in plants, light activated enzyme.
phr gene in E. coli. 2. Excision-repair common
recognition enzyme see damage, then excision of
region that includes the damaged bases, followed
by new DNA synthesis. Multiple systems in a
cell. These handle most of the repair.

6
Types of repair systems 3. Mismatch repair
mismatches that occur doing replication are
corrected by distinguishing between old and new
strands. 4. Tolerance systems allow replication
in case of structural damage accepting high error
rate. Important in eukaryotes. 5. Retrieval
systems recombination-repair, another type of
tolerance system. Corrects by recombining with a
good copy of the damaged region. Mostly found in
bacteria.

7
Major pathways of repair 1. uvr
excision-repair system. 2. dam replication
mismatch-repair system. 3. recB and recF
recombination and recombination-repair pathways.

8
Excision-repair systems in E. coli
Steps in excision repair 1. Mismatch and/or
distortion of structure. 2. Incision
endonuclease cleavages of both sides of damaged
base. 3. Excision 5-3 exonuclease removes DNA
between nicks. 4. Synthesis pol I replaces
damaged region. 5. Ligation ligase seals the
nick.

9

Fig. 14.27
10
Excision-repair systems in E. coli
Uvr system involved in short patch and long
patch repair. 1. endonuclease uvrA, B, and C
cut DNA at positions 7 nt from 5 side and 3-4 nt
on the 3 side of the damaged base (12 nt
total). After cleavage this DNA can act as
substrate for several systems that excise
thymidine dimers including exoVII and DNA pol I
(most important).

11
Excision-repair systems in E. coli
Uvr system 2. Helicase uvrD unwinds the cut
region to release the strand (12 nt therefore
short-patch repair). 3. 99 of repair by uvr
system is short-patch long-patch repair involves
uvrR protein. 4. long-patch repair involves
removal of 1500 to 9000 nt around replication
forks. DNA pol I resynthesizes the strand.

12
Uvr system
UvrA
recognition (uvrA)
UvrB
(Deformed DNA)
Fig. 14.28
UvrB
UvrC
nicking
7 bp 5 3-4 bp 3
helicase exonuclease
DNA pol I
UvrD
5
3
DNA pol I
ligase
5 to 3 exonuclease
13
Excision-repair systems in E. coli
Mutator (mut ) phenotype Other types of repair
systems are high fidelity however Error-prone
DNA replication occurs after UV irradiation.
These mutations were given the name mut. Many
of the genes originally identified have turned
out to be in genes previously identified as
components of replication or repair systems.

14
Excision-repair systems in E. coli
1. The error-prone phenotype may be part of a
tolerance pathway, not just mutations in repair
or replication genes. 2. mutations in the umuD
and umuC genes abolish induction of UV repair.
These genes part of an operon that is induced by
UV. Plasmids carrying homologs of these genes
increase UV resistance to killing, but the price
is increased susceptibility to mutagenesis.

15
Excision-repair systems in E. coli
p. 452 Error-prone activity umuDC operon encodes
proteins that form the UmcD2C complex. UmuD is
cleaved by RecA to form D. The UmcD2C complex
is called DNA pol V. This DNA polymerase can
bypass pyrimidine dimers or other bulky adducts.
This polymerase is induced as part of the SOS
system.

UmuD x2
UmuD x2
UmuC
RecA causes UmuD protein to be cleaved to form
UmuD. This is the activated form (called DNA pol
V).
UmuC
DNA pol V
RecA
16
Controlling the direction of mismatch repair
When two normal bases are mismatched, how does
the cell know which base to change in order to
restore the original state?

replication error
A
C
17
dam methylation system marks the original
strand since it is methylated.

m
GATC
A
T
CTAG
replication error
m
A
In period before site is methylated, new strand
is targeted for repair.
C
mutH, mutL, mutS and uvrD
m
GATC
Strands now indistinguishable to repair system
CTAG
m
18
dam methylation system marks the original
strand since it is methylated.

m
GATC
CTAG
replication error
E. coli dam - strains show high rate of
spontaneous mutations. These are mutations during
replication.
m
A
C
mutH, mutL, mutS and uvrD
m
GATC
A
T
CTAG
Newly synthesized DNA
m
19
MutS
dam methylation mismatch repair
Fig. 14.30
MutL
MutS
MutS translocates to GATC site. Nonmethylated
strand is then selected for excision all the way
back to the damaged site.
MutH
L
S
GATC
Recognition of the GATC site causes Mut H to join
the complex where it acts as an endonuclease to
nick the unmethylated strand.
uvrD
New strand synthesized by DNA pol III.
uvrD unwinds the nicked strand.
20
Default repair systems that show bias in error
correction.
C deaminates to T
VSP system uses MutS/L to remove T from GT and CT
mismatched pairs. Not dependent on GATC site.
G
T
G
C
MutY removes A from CA and GA mismatched pairs.
Does not use the mutS/L system. MutY encodes
Adenosine glycosylase which creates an apurinic
site. This is removed by an excision repair
system.
C
A
C
G
G
A
G
C
21
Retrieval systems in E. coli

Also known as post-replication repair or
recombination-repair.
damage remains
5
T-T
RecA
single-strand exchange
3
retrieval
Fig. 14.31
22
Retrieval systems in E. coli

Role of the recA protein 1. In uvr (excision
repair) mutants, introduction of a mutation in
recA eliminates all remaining repair
capabilities. 2. Replication in uvr -/recA -
double mutants results in production of DNA
fragments whose size corresponds to the distance
between thymidine dimers. More than 1-2
thymidine dimers are lethal. Wild type can
tolerate up to 50.
23
Retrieval systems in E. coli

Activities of the recA protein 1. The recA
protein is involved in normal recombination and
in single-strand exchange of recombination-repair.
2. The recA protein is activated by UV
irradiation and is thought to induce latent
protease activity in its target proteins (LexA
repressor, ? repressor).
Fig. 14.32
24
An SOS system of many genes

Stress conditions that damage DNA or inhibit
replication induce a family of genes that
comprise the SOS response. All of these
stresses initially act upon the recA protein,
which in turn inactivates the LexA
repressor. Induction conditions UV,
cross-linking and alkylating agents, thymine
shortage, and mutations in some dna genes.
(single-stranded DNA and ATP same as required
for RecA function in recombination)
25
The SOS system
SOS repair system includes a by-pass system
(tolerance system) that allows DNA replication
across damaged areas at the cost of fidelity.
SOS repair is a major cause of UV-induced
mutagenesis! The recA protein appears to inhibit
the editing function of DNA pol III by directly
binding to the thymidine dimer. Mismatches in
the opposing strand are not corrected.
26
An SOS system of many genes

Still a mystery regarding the mechanism of recA
activation. Inducer may be an intermediate in
DNA metabolism 1. DNA structure? 2. small
molecule released from DNA? 3. single-stranded
DNA and ATP ? (these are sufficient in vitro)
27
RecA activation of LexA
lexA activated to self-cleave

recA
activated recA
UV
lexA repressor
induction
repair gene
repair gene
SOS box
28
UV
Regulation of uvrB by dual promoters

himA gene
umuC gene
lexA repressor
uvrA gene
uvrB gene
recA gene
Promoter-1 (repressed by lexA)
Promoter-2 (constitutive)
lexA gene
29
UV
Activation of lexA

recA
All targets of recA are cleaved at the dipetide
Ala-Gly. Very little or no other amino acids in
common suggesting that protein structure plays an
important role in target recognition.
lexA
ala-gly
30
Regulation of the SOS system

LexA repressor protein normally keeps levels of
LexA, RecA and excision repair enzymes low
repair genes
lexA
RecA gene
LexA gene
Fig. 16.16
31
by-products of DNA damage
UV

recA
repair genes
lexA
LexA is also induced, but is rapidly inactivated
by RecA.
lexA
RecA gene
LexA gene
32
After damage is repaired, recA is no longer
activated. This results in the build-up of LexA
protein which shuts down the SOS system.

recA
repair genes
lexA
lexA
RecA gene
LexA gene
33
Mammalian repair systems

• Characteristics
• 1. only 3-4 bases replaced (short-patch repair
  only).
• 2. MSH repair system in yeast is homologous to
  the E. coli mut system. This system repairs
  mismatched base pairs.

34
Mammalian repair systems

• Characteristics
• 3. human hereditary disorder xeroderma
  pigmentosum (XP) results in extreme sensitivity
  to sunlight. Explained in terms of a failure in
  excision repair of pyrimidine dimers. Nine
  complementation groups characterized by
  deficiency in excision repair.

35
Mammalian repair systems

• Characteristics
• 4. In yeast the RAD gene products (RAD3, RAD6
  RAD52) are involved in repair of radiation damage
  (UV and others). RAD3 is involved in excision
  repair. One of the genes responsible for XP is
  in the RAD3 group of humans. RAD3 is a helicase
  subunit of transcription factor IIH (TFIIH). Note
  than in eukaryotes DNA repair is closely linked
  to transcription.

36
A common system repairs double-strand breaks
Genes VIII section 15.29
No slides, but read the text and understand this
material pp. 460-462.