UNIT 4
DNA repair

INTRODUCTION
 DNA damage occur during replication
 Can be caused by environmental agents such as radiations, chemicals etc.
 DNA repair systm 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 SUMMARISED
G A T C
ds DNA Break Mismatch
Thymidine dimer
AP site
Covalent X-linking
ss Break
C-U deamination

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
 Photlyase 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
 Role of uvr gene product can be determinde by the mutants
 xeroderma pigmentosum in human is due to inability to carry out excision repair

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 ε 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 cell’s life, the SOS system is turned off, because lexA
represses expression of all the critical proteins.
 However, 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 lexA’s 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.

Gene Repair Function
lexA SOS repressor
recA
SOS regulator; SOS mutagenesis; recF-dependent recombinational repair; recB-dependent repair of double-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
uvrD
Short-patch nucleotide-excision repair; recF-dependent recombinational repair; recB-dependent repair of double-strand
gaps; cross-link repair; methylation-directed mismatch repair
dinA SOS mutagenesis (?)
sulA Inhibitor of cell division
List of some SOS
regulon genes in E. coli

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, MutSα
and MutLα, 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) singlestranded DNA binding protein (SSB)
4) DNA polymerase III holoenzyme
5)DNA ligase
 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

 action of a specific helicase(UvrD) and one of three exonucleases.
 The helicase unwinds the DNA ,starting from the incision and moving
in the direction of the site of the mismatch ,and the exonuclease
progressively digests the displaced nucleotide.
 This action produces a single-strand gap, which is then filled in by
DNA polymeraseⅢ and sealed with DNA ligase.
 Dam methylases tags the parental strand by transient
hemimethylation and methylates A residues on both strands of the
sequence 5’-GATC-3’.
 MutH protein become activated only when it is contacted by MutL
and MutS located at a nearby mismatch
 In human proteins are
1) MutSα and MutLα
2)PCNA and RPA
 human MutS and MutL homologues are heterodimers
 hMutSα preferentially recognizes base-base mismatches