Dhanu DNA Repair

8/2/2019 Dhanu DNA Repair

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DNA REPAIRMECHANISM

Presented byDhananjay N. Gotarkar

ID. No. PAL-0222Dept. of Plant Biotechnology,

UAS, GKVK, Bengaluru


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WHY DNA NEEDS REPAIRMECHANISM ?

DNA is susceptible to damage due to :-

Ionizing radiation breaks the backbone of the

structure

Metabolites alter base structure

UV Radiation adjacent pyrimidines get dimerised

Mutations affect germ cells

modified trait passed on

Mutations affect somatic cells


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Types of DNA DamageSummarized

G A CT

ds DNA Break Mismatch

Thymidine dimer

AP siteCovalent X-linking

ss Break

C-U deaminati


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DNA had -

Huge variety of repair mechanisms to

repair DNA

Proteins patrol DNA searching for

alterations and distortions

Most repair systems excise the

damaged section

TO OVERCOME THIS.


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DNA REPAIR MECHANISMS -

Damage Reversal

Excision Repair

Double Strand Break Repair

Postreplicative Repair


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I. DAMAGE REVERSAL

1-Photo reactivation:

Most simple way for DNA repair : a single step

reaction.

Photolyase enzyme can split pyrimidine dimers:

breaks the covalent bond.

Existence in mammalian not yet proved.

2- Direct DNA Repair :

- Enz me that erform Direct DNA re air is O6-


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Photoreactivation (the enzyme DNAPhotolyase captures energy from light )

Direct reversal of DNA

damage


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II. EXCISION REPAIR

1-Base Excision Repair ( B E R ):

It repairs small, non bulky DNA lesions: methylated,

oxidized, reduced bases. It is estimated to occur 20,000 times a day in each

cell in our body.

Damaged or inappropriate base is removed from itssugar base (Glycosidic bond) linkage and replaced

with correct base.


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1- Removal of the damaged base by a DNA glycosylase. Eight

enzymes, each one responsible for identifying and removing a

specific kind of base damage.

2- Removal of its deoxyribose phosphate in the backbone, producing

a gap: an AP site. Two genes encoding enzymes with thisfunction.

3- Replacement with the correct nucleotide. Done by DNA

polymerase beta ( one of at least 11 DNA polymerases encoded

by our genes ), using the other strand as a template.

4- Ligation of the break in the strand. Two enzymes are known that

can do this, DNA Ligase

Different Steps of BER:


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Q. How the Glycosylases gain the access to theinappropriate or damaged bases within double

helix ? Recently, it has been demonstrated that these

enzymes remove the damaged bases by

flipping them out known as base flipping.

For ex. In human being enzyme 8-oxoguanine

DNA glycosylase, which recognizes 8-oxoguanine and flip the base out to excise it.

Base flipping seems to be common mechanism

in DNA r ir


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2-Nucleotide Excision Repair( N E R ):

The process of NER is biochemically

complicated, 30 distinct proteins that function

as a large complex called the nucleotide

excision repairosome.

It is the most important DNA repair pathway,

The sole repair system forbulky DNA lesions,

which creates a block to DNA replication and

transcription.


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1. UvrA and UvrB scan DNA to identify adistortion (e.g. Thymine dimer)

2. UvrA leaves the complex, and UvrB melts

DNA locally round the distortion3. UvrC forms a complex with UvrB and creates

nicks to the 5 side of the lesion

4. DNA helicase UvrD releases the singlestranded fragment from the duplex, and DNA

Pol I and ligase repair and seal the gap

Steps in NER in E.coli are:


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Fig. Nucleotide

Excision Repair in

Note: UvrA gene are

responsible for UV distortionrepair Protein


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3- Mismatch Repair ( M MR ):

This process occurs after DNA replication as a last "spellcheck" onits accuracy. It encompasses about 99% of all DNA repair.

The mismatch repair system follows behind the replicating fork,

recognizes this mistakes/problems

Like - base mispairs, short insertions and deletions are excised as

single nucleotides by a group of repair proteins which can scan

DNA and look for incorrectly paired bases (or unpaired bases)

which will have aberrant dimensions in the double helix.

Synthesis of the repair patch is done by a DNA polymerase .


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Mismatch Repair in

Note :The genes arecalled mut formutator because itcauses high level ofspontaneousmutation in cell


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III. DOUBLE-STRAND BREAKREPAIR

Naturally occurring reactive oxygen molecules

and ionizing radiation are prevalent sources of

such damage.

DSBs are a major cytotoxic lesion : even a

single unrepaired DSB can be a lethal event.

There are two different mechanisms of repair:

Non Homologous end joining repair

Homology-directed recombination


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1- Non homologous end joining ofDSBs:

Joins broken chromosome ends in amanner that does not depend on sequence

homology and may not be error free. incorrect

ends may be joined, and repair mechanismcauses sequence errors.


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Steps in Non Homologousend joining -

Three steps in end joining repair of DSB's:

1- Recognition of broken ends.

2- Unwinding of short stretch of DNA to uncover short

regions of homology "microhomologies"

3- Removal of unpaired regions and ligation of products.


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2 - Recombination repair:

homologous recombination:

It is an important and preferred mechanism ofrepair since it is least likely to result in

mutations:

broken ends are repaired using the information

on the intact homologous chromosome.


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IV. POST REPLICATIVE REPAIR

Error prone repair: Involves the replication machinery bypassing sites of base damage,

allowing normal DNA replication and gene expression to proceed

downstream of the (unrepaired) damage.

Error prone polymerases are developed by evolutionary processes.

Involves low-fidelity DNA polymerases that are able to bypass DNA

lesions that stall the high-fidelity polymerases required for DNA

replication.

To overcome the block, these 'sloppy copiers' add nucleotides to the

replicating strand opposing the DNA lesion,

- allowing replication to continue,

- and introducing mutations into the newly synthesized sequence.


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PREVENTING DNA DAMAGEAND

IMPROVING THE CAPACITY OF DNA REPAIR:

These two goals seem possible by:

1. Insuring enough intake of antioxidants as vitamins

C and E and beta-carotene. 2. Caloric restriction ( reduction of total daily calorie

intake by about 35% for animals).

3.Avoiding exposure to UV and ionizing radiations,and toxic chemicals.

4. Fighting infections


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INFERENCE

DNA: many kinds of damage: base damage,

mispairing, single or double strand break

Many repair mechanisms:- Damage reversal (Photoreactivation,

ligation)

- Damage removal: BER, NER, and MMR

- Damage tolerance: end joining repair,

recombination repair, and error prone repair.


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REFERENCES

Woods C G. DNA repair disorders. Arch Dis

Child 1998;78:178-184.

Friedberg E C. DNA damage and repair.

Nature 2003;421:436-440.

Wood R D. and al. Human DNA repair genes.

Science 2001 Feb 16;291:1284-1289.

Principles of Genetics Robert H Tamarin

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