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L15 DNA Repair

Table 5-2 Molecular Biology of the Cell ( Garland Science 2008)


5-Methylcytosine is a hotspot for


mutation

Microsatellite DNA- microsatellite DNA are mutation- prone sequences of up to 10 base


repeats, for example CACACACA-homologous bonding is error prone causing
recombination slippage, reducing or duplicating the number of repeats thus
microsatellite length is highly variable.

Photoreac)va)on A photodimer may be


reversed by direct repair involving 300nm light.
In its absence.other repair pathways.

Other specific repair enzymes :Alkyl and methyl transferases


also reverse specific lesions

E coli has 5 DNA polymerases.





DNA pol III -
replicase with 3-5 proofreading

DNA pol I -
major repair -5-3 exonuclease activity enables
it to start replication at a nick (+ 3 - 5 proofreading)

DNA pol II -
minor, SOS repair

DNA pol IV, V
SOS repair.



DNA polymerase I,II,IV, V are in high copy number
compared to DNA pol III and are mostly involved in repair



Many repair systems use complementarity, antiparallel
complementarity to guide repair homology dependent repair

Faithful, but not perfect short term replication checks



Copy errors in replication could cause heritable changes to a DNA
sequence.

There are 3 error - checking steps in DNA replication:

(1) base selection (2) proof reading (3) mismatch repair

(1) Base selection if the correct base is not chosen, the polymerase
site in the holoenzyme will not be activated.

(2) Proof reading: 3-5 exonuclease activity (DNA pol III)

(3) Mismatch repair after replication, the wrong nucleotide will
not generally pair correctly, it causes a bulge in the helix,
mismatch repair enzymes (Mut S, L and Mut H) scan for bulges, if
found soon after replication, non-methylated strands are repaired
based on its methylated complement. See fig 15-26

If both strands are methylated, the mutation may be inherited.

Eukaryotes & prokaryotes: A replication (or RNA expression) fork


that is blocked by damage to several bases signals the cell death
pathway, But it may be repaired


It may be repaired just before replication by:






(1) Nucleotide Excision Repair (NER) involving a complex of
proteins that:

-Recognition of a distorted double helix
UVr A dimer, B

-Cuts on both sides of the damaged strand
UVr B, C

-Removes the damaged segment
UVr D

-Restore using DNA polymerase I (fills), and DNA ligase (links)
the new segment to the strand.


During replication E coli- SOS repair use DNA polymerase II, IV,
V (eukaryotes - translesion repair). These are low - fidelity (error
prone or high mutation) systems involving recombination-like
repair, that: 1) puts an undamaged strand near the damaged one;
2) excise the damaged part , then; 3) copy undamaged.


SOS repair in prokaryotes &


Translesion DNA synthesis in
eukaryotes.

Replication blocks may signal cell
apoptosis, but the cell may survive
if it enables a different class of
polymerases called translesion or
bypass polymerases that have a
much higher error rate (lack
3-5 proof reading) than the main
DNA polymerases, and only
synthesize few bases then
detach

Ub ubiquitin binds bypass


unwind

UV induced
Rec A binds
SSB, binds
single and
double strand
DNA.
Inducessignals

Bypass Poly
V-PCNA

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

Two pathways for nucleotide-excision repair


Recognize

TFIIH 10 subunits

Cut -nuclease excision



Unwind with a helicase (2 subunits
XPB,XPD)

Remove
and stabilize with


single stranded DNA
binding protein RPA



Restore with bypass polymerase

And DNA ligase

Large number of
other proteins act to
remove damaged
base + 30 others..

Characteristics of lesion bypass


polymerases

Error rate 100-10,000 x higher on undamaged
templates

Lack 3 to 5 proofreading exonuclease
activity

Exhibit distributive (bind, add a few
nucleotides , then dissociate) rather than
processive polymerization (# nt. incorporated
per binding unbinding at the end)

Support translesion DNA synthesis in vitro

Mismatch repair (MMR)



Despite extraordinary fidelity of DNA synthesis, errors do
persist

Such errors can be detected and repaired by the postreplication mismatch repair system. MMR also processes
mispairs that result from heteroduplex DNA formed
during genetic recombination: act to exclude
homeologous (partly homologous) recombination

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)

Mismatch repair corrects


replicative errors

Transition mispairs G:T and A:C and


one base loops are particularly wellrecognized (these are also the most
common polymerase errors) and repair is
targeted to the newly synthesized
strand without adenine methyl tags

Post Replicated DNA


Photo and other damage


A homology-dependent base-excision repair


in post-replicated DNA

Recognition Each glycosylase recognizes a
specific type of altered base (oxidized,
deaminated etc..)

(a) Cleave bond Glycosylase cleaves the
glycosidic bond, (base-sugar), leaving
unattached bases

(b)Cut AP endonuclease(s) (APurinic,
APyrimidinic), nicks the damaged strand

(c)Remove deoxyRibophosphodiesterase
(dRpase) removes neighbor bases

(d) Restore DNA polymerase I and DNA
ligase.

Induced
mutations
through the action of
mutagens.



High energy like X rays
cause double strand
breaks among various
other mutations


Measured as % death of F2
(hemizygous) males carrying
irradiated , P, X chromosomes
through marked , heterozygous F1
females

4


Cause:


Induced
mutations
through the action of
mutagens.



High energy like X rays
cause double strand
breaks among various
other mutations


Measured as % death of
(hemizygous) males

Double Strand Break Repair



(1)Non Homologous, End Joining
(NHEJ) mechanisms involve
dedicated proteins repairing
non-replicating chromosomes
in G0 or 1 (pp540)



(2)Synthesis-Dependent Strand
Annealing (SDSA) mechanism
uses a sister chromatid as a
template (homologous
recombination repair) G2,
mitosis.

(3) Meiosis-mechanisms: break
and repair using homologues.

Error-prone
nonhomologous end
joining repairs double

strand breaks causes minor
mutations G1, (S or G2):



Bind Ku80- Ku 70

Trim

Ligate

Figure 5-51 Molecular Biology of the Cell ( Garland Science 2008)


Error-free repair of double


strand breaks by Synthesis
Dependent Strand
Annealing (SDSA)
using the sister
chromatids available (in
mitosis) as a template for
repair.

(1) Trim 5 ends with an
exonuclease, and

(2) Coat single strands
with a Rad51 (recA
homologue).

(3) Strand invasionsearch for sister chromatid.

(4) Free 3OH primes DNA
synthesis, unwind, anneal
and seal with DNA ligase

Replication fork

assembles on
Displacement
loop

Crossing over during meiosis is a


frequent cause of mutations

Meiotic Recombination

Double-strand CUTS (SpoII)
initiate meiotic recombination,
binds to free 5 ends and protects
from free binding

(1) Attracts proteins to trim 5 end
and unwind,

(2) Rad51attaches to the 3 end.

(3) Coats strand and associates
with Dmc1-Rad.

(4) Rad51-Dmc-1 scans for
complementary sequence in a
homologous chromosome.

(5) Strand invasion and chiasma
formation - D loop strand
extension and ligation