MECHANISMS OF DNA REPAIR

Dr Radhouane GOUIAA Policlinique CNSS 3018 Sfax TUNISIA

5th ADVANCED POSTGRADUATE COURSE 2nd Training Session June 2003

DNA DAMAGE
1- Tautomerisation: spontaneous Guanine: Keto form: pairs with Cytosine Thymine: Keto and Enol forms Adenine and Cytosine: Amino and Imino forms
Enol form: mispairs to Thymine

1- Tautomerisation: 2- Deamination: spontaneous base degradation Base C A G Pairing G T C Deamination U Hypoxanthine Xanthine Mispairing A C C

3- Oxidation:damaging bases by free radicals of oxygen 4- Alkylation: addition of alkyl groups to bases or backbone DNA 5-Depurination: loss of Adenine or Guanine bases: 10 000 bases per day per cell Other DNA hydrolysis reactions 6-Pyrimidine dimers: by sunlight ( UV ). 7-Single or double strand break.

DNA REPAIR MECHANISMS

Three kinds:

A-Damage reversal B-Damage removal C-Damage tolerance

A-Damage reversal
may be divided in three different kinds:

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.

A-Damage reversal
1- Photo reactivation:

2-Most of damaged bases are repaired by

one enzyme, removes mismatched base restoring the correct one. Without the need to break the DNA backbone: - Uracil, product of cytosine deamination, is detected, removed by uracil N-glycosylase, and replaiced by cytosine.

-Hypoxanthine, product of adenine deamination, is recognized, removed by hypoxanthine Nglycosylase then replaced by adenine -Alkylation is repaired by enzymatic transfer of methyl group by Methyl Guanine-DNA MethylTransferase ( M G M T ), and we will have Guanine -Demethylation is used to repair 1-methyl adenine and 3-methyl cytosine.

A-Damage reversal
1- Photo reactivation:

2-Most of damaged bases

3-Ligation of simple strand breaks:
Simple breaks in one strand are repaired by DNA ligase.

A-Damage reversal B-Damage removal:

Is the most common repair mechanism. It includes: base excision repair, nucleotide excision repair, and mismatch 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 linkage and replaced.

Different steps of BER: a- removal of the damaged base by a DNA

glycosylase. Eight enzymes,each one responsible for identifying and removing a specific kind of base damage.

b- removal of its deoxyribose phosphate in the

backbone, producing a gap: an AP site.Two genes encoding enzymes with this function.

Different steps of BER: c- 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.

d- ligation of the break in the strand. Two

enzymes are known that can do this.

1-Base excision repair ( B E R ):

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. - The most important DNA repair pathway, - The sole repair system for bulky DNA lesions, which creates a block to DNA replication and transcription. - Can also repair many of the same defects that are corrected by direct repair, base excision and mismatch repair

( N E R ):

It probably recognizes lesions that distort the DNA

Steps in NER are:

a-

Recognition of damage by one or more protein factors. b- Assembly of repair complex: nucleotide excision repairosome. c- Double incision of the damaged strand several nucleotides away from the damaged site, on both sides, by an endonuclease.

( N E R ): dRemoval of the short segment ( about 24 to 32 nucleotides ) containing the damaged region, by an exonuclease. e- Filling in of the resulting gap by a DNA polymerase: synthesizes DNA using the opposite strand as a template. f- Ligation: a DNA ligase binds the synthesized piece into the backbone.

1-Base excision repair ( B E R ):

2-Nucleotide excision repair ( N E R ): 3- Mismatch repair ( M M R ):

This process occurs after DNA replication as a last "spellcheck" on its accuracy.

( M M R ):
Incorrect bases incorporated as a result of mistakes during DNA replication ( 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 .

A-Damage reversal B-Damage removal

C-Damage tolerance
It is not truly repair but a way of coping with
damage:

C-Damage tolerance:
1- Double strand break ( D S B ):
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:

1- Double strand break: a- End joining repair 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.

a- End joining repair of DSBs:

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. This mechanism: called non homologous endjoining.

a- End joining repair of DSBs:

b- 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.
Requires an extensive region of sequence homology between the damaged and template strands.

C-Damage tolerance:
1- Double strand break ( D S B ): a- End joining repair of DSBs:
b- Recombination repair: 2- Error prone repair:
It is used when all else fails + + +

2- 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.
Involves low-fidelity DNA polymerases that are able to bypass DNA lesions that stall the high-fidelity polymerases required for DNA replication.

2- Error prone repair:

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.

SEQUENTIAL EXPRESSION OF REPAIR SYSTEMS:

- Damage reversal

- Excision-resynthesis ( E B R, then N E R, then MMR)

- Recombination - And finally error prone repair.

DNA DAMAGE, DNA REPAIR, AND AGING:

DNA damage, particularly oxidative lesions, is thought to contribute to aging. In fact, some scientists believe : accumulation of uncorrected DNA damage over years is a major cause of aging.

following observations: * Animals with fastest rates of DNA repair have longest life spans. * Animals with highest rates of oxidative damage by free radicals generally have shortest life spans. * In lower life forms, anti-oxidant supplements, which can correct and prevent DNA damage, increase life span. * Exposure to external causes of DNA damage decreases life span. * Humans who have genetic diseases resulting in greater spontaneous DNA damage or inefficient DNA repair often show signs of premature aging.

PREVENTING DNA DAMAGE AND IMPROVING THE CAPACITY OF DNA REPAIR:

These two goals seem possible by: 1insuring enough intake of antioxidants as vitamins C and E and beta-carotene. 2caloric restriction ( reduction of total daily calorie intake by about 35% for animals ). 3avoiding exposure to UV and ionizing radiations, and toxic chemicals. 4fighting infections

CONCLUSION

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. These mechanisms are not always error free DNA damage contribute to aging Preventing DNA damage and improving capacity of DNA repair may delay aging.

THANK YOU

REFERENCES
1) Troen BR. The biology of aging. Mt Sinai J Med 2003 Jan;70(1):3-22. 2) de Boer J. and al. Premature aging in mice deficient in DNA repair and transcription. Science 2002 May 17;296(5571):1276-9. 3) Woods C G. DNA repair disorders. Arch Dis Child 1998;78:178-184. 4) Friedberg E C. DNA damage and repair. Nature 2003;421:436-440. 5) Wood R D. and al. Human DNA repair genes. Science 2001 Feb 16;291:1284-1289.