Dna Repair Mechanism: ID. No. PAL-0222 Dept. of Plant Biotechnology, UAS, GKVK, Bengaluru

DNA REPAIR MECHANISM
5/7/12
Presented by Dhananjay N. Gotarkar ID. No. PAL-0222 Dept. of Plant Biotechnology, UAS, GKVK, Bengaluru
WHY DNA NEEDS REPAIR MECHANISM ?
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
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Mutations affect somatic cells
Types of DNA Damage Summarized

G ds DNA Break A T C Mismatch
C-U deaminatio
ss Break Thymidine dimer Covalent X-linking AP site
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TO OVERCOME THIS. DNA had Huge variety of repair mechanisms to
repair DNA
Proteins patrol DNA searching for
alterations and distortions

Most repair systems excise the
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damaged section
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
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-
DNA Repair :
Enzyme that perform Direct DNA repair is O6-
Direct reversal of DNA damage
Photoreactivation (the enzyme DNA Photolyase captures energy from light )

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II. EXCISION REPAIR

Is the most common repair mechanism. The general mechanism that works by removing the damaged portion of a DNA molecule. Various enzymes can sense damage or Distortion of DNA double helix. During excision repair, Bases and Nucleotides are removed from the damaged strand. It includes:
5/7/12 Base
excision repair,
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 its sugar base (Glycosidic bond) linkage and replaced with correct base.
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Different Steps of BER:

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 this function. 3Replacement 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
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Q. How the Glycosylases gain the access to the inappropriate 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 8oxoguanine and flip the base out to excise it. Base flipping seems to be common mechanism 5/7/12 in DNA repair.
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 for bulky DNA lesions, which creates a block to DNA replication and
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transcription.
Steps in NER in E.coli are:

1. UvrA and UvrB scan DNA to identify a distortion (e.g. Thymine dimer) 2. UvrA leaves the complex, and UvrB melts DNA locally round the distortion 3. UvrC forms a complex with UvrB and creates nicks to the 5 side of the lesion 4. DNA helicase UvrD releases the single stranded fragment from the duplex, and DNA Pol I and ligase repair and seal the gap
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Note: UvrA gene are responsible for UV distortion repair Protein
Fig.
Nucleotide 5/7/12 Excision Repair in
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3- Mismatch Repair ( M M R ):
This process occurs after DNA replication as a last "spellcheck" on its 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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Note : The genes are called mut for mutator because it causes high level of spontaneous mutation in cell
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Fig. Showing the
Mismatch Repair in
III. DOUBLE-STRAND BREAK REPAIR
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 of DSBs:
Joins broken chromosome ends in a manner that does not depend on sequence homology and may not be error free. incorrect ends may be joined, and repair mechanism causes sequence errors.
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Steps in Non Homologous end 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 of repair 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 DAMAGE AND 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.
5/7/12
Principles of Genetics Robert H. Tamarin

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DNA REPAIR MECHANISM

WHY DNA NEEDS REPAIR MECHANISM ?

DNA is susceptible to damage due to :

modified trait passed on

Mutations affect somatic cells

Types of DNA Damage Summarized

ss Break Thymidine dimer Covalent X-linking AP site

TO OVERCOME THIS. DNA had Huge variety of repair mechanisms to

alterations and distortions

DNA REPAIR MECHANISMS

Most simple way for DNA repair : a single step reaction.

Enzyme that perform Direct DNA repair is O6-

Direct reversal of DNA damage

Photoreactivation (the enzyme DNA Photolyase captures energy from light )

II. EXCISION REPAIR

II. EXCISION REPAIR

1-Base Excision Repair ( B E R ):

Different Steps of BER:

2-Nucleotide Excision Repair ( N E R ):

Steps in NER in E.coli are:

Note: UvrA gene are responsible for UV distortion repair Protein

Nucleotide 5/7/12 Excision Repair in

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

Fig. Showing the

III. DOUBLE-STRAND BREAK REPAIR

Non Homologous end joining repair Homology-directed recombination

III. DOUBLE-STRAND BREAK REPAIR

1- Non homologous end joining of DSBs:

Steps in Non Homologous end joining

Three steps in end joining repair of DSB's:

III. DOUBLE-STRAND BREAK REPAIR

IV. POST REPLICATIVE REPAIR

Error prone repair:

PREVENTING DNA DAMAGE AND IMPROVING THE CAPACITY OF DNA REPAIR:

These two goals seem possible by:

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.

Principles of Genetics Robert H. Tamarin

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