Presenter: PARICHAY S.R II SEM APPLIED GENETICS 14KUSAG004

CONTENTS

INTRODUCTION. MISMTACH REPAIR. RECOMBINATIONAL REPAIR. PHOTOREACTIVATION REPAIR. SOS REPAIR. ROLE OF P53 IN DNA DAMAGE AND APOPTOSIS. SUMMARY.

INTRODUCTION

DNA Mismatch Repair is a system for recognising and repairing erroneous insertion, Deletion and misinconcorporation of bases. That can arise during DNA replication and recombination. It is Strand Specific

Normally inside the DNA we have base pairing called – Watson crick base pairing i.e A T G C If U present its dangerous because it is formed due to Deamination of C Cytosine deamination Uracil

Require 3 proteins called MUTATOR CONDUCTIVE PROTEINS (MUT proteins). 1) MUTs 2) MUTl 3) MUTh Other requirements: uvrD, DNA pol I, Ligase.

U

U

S

U

S L

U

S

L

STEP 1:

STEP 2:

STEP 3:

U

L

H

U

U

STEP 4:

STEP 5:

STEP 6:

STEP 7:

MUTs recognise that region where incorporation of wrong nucleotide occurs. But MUTs cannot differentiate b/n parental & daughter strands. MUTl will bind to methylated region i.e parental strand (hence help in differentiating) Once recognised MUTl also created an area for daughter strand to cleave( It forms loop like structure bringing everything together), marking misincorporation region.

MUTh has vital activity of cleavage(making a nick), helps in cleaving the uracil from Backbone. Even though the uracil has been separated it is bonded by hydrogen bonds, uvrD Removes it. DNA polyIII Will further elongate the Strands.

Recombinational repair is a way of using one copy of cells Information to ensure that overall information store remains Intact.

5I 3I Step 1 Coating of Single Strand with RecA.

Step 2 Synapsis with DNA duplex.

Step 3 Alignment at homologous sequences

Step 4 Formation of Joint molecule.

Step 5 Branch migration.

ATP ADP+Pi 3I

3I

5I

5I

RecA protein

The biochemical process of recombination occurs by breaking and rejoining of DNA strands. The key of reaction is strand displacement initiated at a nick in the chromosome. RecA binds to single stranded DNA and catalyse its exchange with same sequence of Duplex. RecA will cover an entire, single stranded DNA molecule rather than binding to several molecules partially.

The Reaction of RecA coated DNA is the movement of the single stranded regions of DNA to form Joint molecule -process called Strand displacement. In Homologous recombination, two double helices align and are nicked, then RecA catalyses the Inversion of each double helix -- the crossed structure is called Holiday junction.

G C T G T T G G T A

C G A C A A C C A T

G C T G

T = T

G G T A

C G A C A A C C A T

Ultraviolet light

Thymidine dimer

G C T G

T = T

G G T A

C G A C A A C C A T

G C T G

T = T

G G T A

C G A C A A C C A T

Visible light

G C T G T T G G T A

C G A C A A C C A T

UV radiation causes pyrimidine Dimer b/n adjacent pyrimidine. These dimer causes kink in the strand on which they occur, disrupting the Replication of DNA at that point. DNA Photolyase recognise the kink in the DNA and bind to its

site.

When excited by blue light, the Photolyases change the conformation breaking the Dimer,

In Bacteria: DNA photolyases, coded by phr genes, allow them to repair the thymidine dimers caused by UV exposure. Here Folate is used to harvest light energy, which is quickly Transduced to FADH making it FADH2. Now electronically excited FADH2 can transfer high energy electron To dimer causing the structure to break apart into separate thymine Molecules.

The SOS Response is a global response to DNA Damage in which the cell Cycle is arrested and DNA repair and mutagenesis is initiated. The System involves RecA protein, The RecA Protein, stimulated by single stranded DNA is involved in Inactivation of lexA repressor, thereby inducing the response.

During normal growth, SOS genes are negatively regulated by lexA repressor dimers, lexA binds to 20bp sequence in the operator region of those genes. Activation of SOS genes occur after DNA damage by the accumulation of single stranded regions, generates at replication forks, where DNA polymerase is blocked.

RecA forms a filament around these SSDNA’S region in a ATP dependent fashion and becomes activated. The activated form of RecA interacts with lexA repressor to facilitate the lexA repressor self cleavage from operator. Once the pool lexA repressor is reduced causes coordinate depression of SOS gene. By this Damage is repaired and RecA releases DNA.

THE SOS RESPONSE TO REPLICATION DAMAGE

Replication stall and DNA Damage

RecA Activation

lexA autocleavage

The SOS Genes

LexA Derepression

DNA Damage Repair

It is 53kd tumour suppressor protein that prevents cell cycle progression or induces apoptosis in cells with damaged DNA. More than 50% of Cancers are known to have mutation in P53 genes that encodes P53 protein. Master Guardian and Executioner.

Damaged DNA activates ATM protein kinase leads to phosphorylation of P53 protein. P53 leads to transcription of MDM2(ubiquitin protein ligase) which is a CDK inhibitor, prevents the phosphorylation of Retinoblastoma protein leading to cell cycle arrest.

Whenever there is a DNA Damage, there is a rise in amount of P53 gene inside the cell -- this P53 will trigger another protein called BAX. This BAX protein has significant role in mitochondria, there is formation of channel called BAX channel. BAX channel will trigger the release of Cytochrome C This Cytochrome C release induce the production of Caspase 9 : apoptotic executioner, the caspase 9 will induce cell to undergo Apoptosis.

P53

P53

P53

P53

BAX

CASPASE 9

BAX Channel

DNA Damage

Cytochrome release

http://www.cliffsnotes.com/sciences/biology/biochemistry-ii/dna-structure-replication-and-repair/dna-recombination-and-repair

http://www.sciencemag.org/content/291/5507/1284.full An Introduction to Genetic Analysis-Griffith. Youtube channel: Soma’s Biology.