UNIT 4

DNA repair
 INTRODUCTION
DNA damage occur during replication
Can be caused by environmental agents such as radiations, chemicals etc.
DNA repair system is not that much efficient
If it was perfect no evolution would have happened
Three mechanisms alter DNA structure

i) base substitutions during replication

ii) base change due to chemical insteability of bases

iii) action of other chemicals and environmental factors

 EFFECTS OF THE

MECHANISMS
Mismatch - deamination of cytosine to uracil
Depurination N Glycosidic bond spontaneously broken down at
physiological temperature

- 1 purine per 300 purine is removed

UV induced dimer formation T-T
SS breaks phosphodiester bond break by peroxides
DS breaks
Cross linking antibiotics (mitomycine C) or reagents like nitrite ion form
covalent linkages base in one strand and base in other strand
MISMATCH

06_23_DEPURINATION.JPG
06_25_MUTATIONS.JPG
 TYPES OF DNA DAMAGE SUMMARIZED

G A T C

ds DNA Break Mismatch

C-U deamination

ss Break
AP site
Covalent X-linking
Thymidine dimer
 MECHANISMS OF REPAIR
2 major classes

i) light induced repair

ii) light independent pathways

Photoreactivation involved in the first class
Latter consists of

i) excision repair

ii) recombination repair

iii) SOS repair

iv) Mismatch repair

 PHOTOREACTIVATION
Enzymatic cleavage of thymine dimers
Activated by visible light ( 300 - 600nm )
PR enzyme / photolyase
1st enzyme binds to T-T specifically
When light absorbed T-T will be monomerised
Photolyase releases when repair completed
 EXCISION REPAIR
Multi step enzymatic process
2 mechanisms

a) Incision step

b) Breakage of N- glycosidic bond of T-T

) INCISION STEP
) In E.coli repair endonuclease recognises distortion produced by T-T
) Makes 2 cuts in sugar-phosphate back bone
) 1st at 8 bp to 5 and 2nd one at 4-5 bp to 3
) 5 incision produce a 3 OH
 Pol I use that 3 OH as primer to synthesise new strand
This new strand will replace the dimer containing strand
Joining of newly synthesised strand to original strand by ligase
Excised strand will be degraded to single nucleotides by the scavenging exo
and endo nucleases
BREAKAGE OF N- GLYCOSIDIC BOND OF T-T
1st step - enzymatic cleavage of N- glycosidic bond in the 5 thymine
nucleotide of dimer
2nd step is the endonuclease activity of the same glycosylase enzyme to
recognize a deoxyribose lacking a base
Make a single cut at 5 of T-T
 Causes formation of 3 OH
Since it is on a base pair free deoxyribose it can not be used to prime DNA synthesis
In an unknown way deoxyribose will be removed and Pol I act at the new 3 OH
Strand displacement in an excision step by Pol I
Filling the gap
Mammalian mechanism is poorly understood
In E.coli incision is determined by products of the following genes

a)uvrA

b)uvrB

c)uvrC

d) uvr D
Other enzymes are DNA pol and ligase
Role of uvr (ultraviolet repair)gene product can be determinde by the mutants
xeroderma pigmentosum in human is due to inability to carry out excision repair
IN E.COLI
Steps
Binding of trimer protein(2 uvr A and uvr B)
Trimer move along DNA scanning for damage
At the T-T trimer stops
Relaese of uvr As
uvrB remain attached to T-T
uvrC bind to the damaged strand and makes 2 cuts at the damaged
strand
uvrD a helicase that bind to the strand and unwinds the damaged
strand from rest
All uvrB,uvrC,uvrD releases and that causes a gap
Strand replacement by DNA pol and ligase
 RECOMBINATION REPAIR
Thymine dimers are produced in large numbers so that it can not be completely
removed by excision repair
Another mechanism for uv induced thymine dimer removal is the recombination
repair
recA gene is involved
During replication thymine dimer causes distortion
Adenine will be added and removed continously
DNA synthesis restarts in 2 ways in such cases

i) postdimer initiation

ii) transdimer synthesis

Postdimer initiation is responsible for recombination repair
Transdimer synthesis results in SOS repair
 Thymine dimer will be excised but gap will not be filled
So daughter strand will be a fragmented one
It can overcome by a recombination mechanism called sister strand
exchange
Good strand from a homologous DNA is excised and used to fill the gap
DNA Pol I and ligase involved in the process
As the strand is not synthesised newly to remove the gap it is less time
consuming and hence much important
As it occur after replication it is called as postreplicational repair
Another name daughter strand gap repair
 SOS REPAIR
Global response to DNA damage in which the cell cycle is arrested and
DNA repair and mutagenesis are induced
Functional recA gene is needed
RecA protein binds to the SS DNA
Binds at distortions
RecA binds with the part of polymerase which is involved in the editing and inhibits
editing function- mutations will persist in daughter strand
Other 2 genes involved are umuC and umuD
Their function is not known
3 hypothesis are there

1) facilitate binding of RecA to the small distortions

2) facilitate binding of polymerase to the distortions

3) enable release of polymerase

 During normal growth, the SOS genes are negatively regulated by LexA
repressor protein dimers
During a normal cells life, the SOS system is turned off, because lexA
represses expression of all the critical proteins.
When DNA damage occurs, RecA binds to single-stranded DNA (single-
stranded when a lesion creates a gap in daughter DNA).
As DNA damage accumulates, more RecA will be bound to the DNA to
repair the damage.
RecA, in addition to its abilities in recombination repair, stimulates the
autoproteolysis of lexAs gene product .
That is, LexA will cleave itself in the presence of bound RecA, which
causes cellular levels of LexA to drop, which, in turn, causes coordinate
derepression (induction) of the SOS regulon genes.
 As damage is repaired, RecA releases DNA; in this
unbound form, it no longer causes the autoproteolysis of
LexA, and so the cellular levels of LexA rise to normal
again, shutting down expression of the SOS regulon
genes.

Activation of the SOS genes occurs after DNA damage

by the accumulation of ssDNA regions generated at
replication forks, where DNA polymerase is blocked.
 List of some SOS
regulon genes inE. coli
Gene Repair Function

lexA SOS repressor

SOS regulator; SOS mutagenesis;recF-dependent recombinational repair;recB-dependent repair of double-

recA
strand gaps; cross-link repair

recN recF-dependent recombinational repair; repair of double-strand gaps

recQ recF-dependent recombinational repair

ruv recF-dependent recombinational repair

umuC SOS mutagenesis (Error prone repair)

umuD SOS mutagenesis (Error prone repair)

uvrA Short-patch nucleotide-excision repair; long-patch nucleotide-excision repair; cross-link repair

uvrB Short-patch nucleotide-excision repair; long-patch nucleotide-excision repair; cross-link repair

Short-patch nucleotide-excision repair;recF-dependent recombinational repair;recB-dependent repair of double-

uvrD
strand gaps; cross-link repair; methylation-directed mismatch repair

dinA SOS mutagenesis (?)

sulA Inhibitor of cell division

 METHYL DIRECTED MISMATCH
REPAIR
Highly conserved biological pathway
Important in maintaining genome stability
It is mainly to repair base- base mismatches and insertion/deletion of
bases during replication and recombination
Inactive mismatch repair will cause spontaneous mutation
Mismatch repair prevent mutagenesis and tumorogenesis
E.coli MutS and MutL and their eukaryotic homologs,
MutS and MutL, respectively, are key players in MMR-
associated genome maintenance
 In E.coli Proteins involved are

1) MutS, MutL, MutH, DNA helicase II (MutU/UvrD),

2) four exonucleases (ExoI, ExoVII, ExoX, and RecJ)

3) single stranded DNA binding protein (SSB)

4) DNA polymerase III holoenzyme

5)DNA ligase
Dam methylases tags the parental strand by transient
hemimethylation and methylates A residues on the sequence
5-GATC-3
Methylation of newly synthesised strand occurs slowly so that
it can be distinguished from old strand
DNA with only one strand methylated is called
hemimethylated
 MutS recognizes base-base mismatches
MutL interacts physically with MutS, enhances mismatch
recognition, and recruits and activates MutH
MutH, an enzyme that causes an incision or nick on one strand
near the site of the mismatch ie; at methylated site
MutH protein become activated only when it is contacted by
MutL and MutS located at a nearby mismatch (DNA looping
mechanism)
MutH protein causes a cut in non methylated strand
 Exonuclease digest the non methylated strand from
cleavage site to mismatch site
Gap will be formed in daughter strand which is filled
by the action of DNA pol III and sealed by ligase.
Mismatch will be replaced by correct strand
In human proteins are

1) MutS and MutL

2)PCNA (Proliferating cell nuclear antigen) and

RPA(Replication protein A)
human MutS and MutL homologues are heterodimers
hMutS preferentially recognizes base-base
mismatches

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ethyl-directed_mismatch_repair.html
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/nucleotide_excision_repair.html