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

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

EFFECTS OF THE MECHANISMS

06_23_DEPURINATION.JPG

MISMATCH

06_25_MUTATIONS.JPG

TYPES OF DNA DAMAGE SUMMARISED

G A CT

ds DNA Break Mismatch

Thymidine dimer

AP siteCovalent 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

recASOS 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

uvrDShort-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