Repair.ppt

Explains DNA repair mechanism.

1. DNA Repair Dr. N.Banu Associate Professor VISTAS

2. DNA REPAIR Alterations of DNA molecules: Base substitutions during replication. Base changes due to instability of the base or of the N- glycosylic bond and Due to chemicals and environmental agents. These mechanisms are responsible for the following defects: An incorrect base in one strand that cannot form hydrogen bond with the corresponding base in the other stand.( i ) failure of editing function or (ii) spontaneous loss of amino group, converting c to u or A to hypoxanthine. Missing bases: Depurination – loss of purine from DNA. Altered bases: Bases are converted to diff. compounds by chemical and physical agents. E.g. β-particles from radioisotopes and X-rays. E.g. thymine dimers .

3. DNA damage 4. single – strand breaks: A variety of agents can break phophodiester bonds. E.g. peroidases , sulfhydryl compounds( cysteine ) and metal ions such as Fe 2+ and Cu2+. Repaired by DNA ligase . 5. Double – strand breaks: If a DNA molecules receives a large no. of randomly located single-stranded breaks, two breaks may be opp. One another, makes break of the double helix. 6. Cross-linking: some antibiotic ( mitomycin C) and some reagents (nitrite ion) form covalent linkages.

4. Types of DNA Damage Summarised G A C T ds DNA Break Mismatch Thymidine dimer AP site Covalent X-linking ss Break C-U deamination

5. REPAIR OF INCORRECT BASES Addition of incorrect base by Pol.I and III, cannot form hydrogen bond with template. This by corrected by editing function. This is called mismatch repair. Deamination : cytosine loses an amino group forming uracil . After one round of replication lead to the replacement of G.C pair by A.U pair, which would become an A.T pair after another round of replication. 1. Removal of the U by Uracil N- glycosylase . It cleaves the N- glycosylic bond and leaves the deoxyribose in the backbone. 2. AP endonuclease (AP – apurinic acid) makes a single cut, freeing one end of the deoxyribose . This is followed by removal of deoxyribose and adjacent nuclotides ( endonuclease activity of Pol.I ) after which Pol.I fills the gap with correct nuclotides .

6. REPAIR OF THYMINE DIMERS 1. Light – induced repair ( photoreactivation ) 2. light independent repair ( dark repair) i . excision of the damaged base (excision repair) ii. Reconstruction of a functional DNA molecule from undamaged fragment ( recombinational repair) and Iii. Disregard of the damage (SOS repair)

7. PHOTOREACTIVATION It is an enzymatic cleavage of thymine dimers activated by visible light (300-600 nm)/. Photoreactivation or PR enzyme is isolated from bacteria, animals. It does not absorb light, not bind to any light absorbing compound.

8. Dark repair Excision repair: It is a multistep enzymatic process. In E. coli – cut-patch-cut-seal. 1. Incision step – repair endonuclease recognizes the distortion produced by a thyamine dimer and makes a single cut in the sugar- phosphate backbone ahead of the dimer . At the incision site there is a 5’P group on the side of the cut containing the dimer and a 3’OH on the other side. 2. The 3’OH is recognized by Pol.I which synthesize new stand while displacing a DNA segment consisting of about 20 nucleotides and carrying the thymine dimer . 3. This segment if excised by 5’-3’ exonuclease activity of Pol.I . 4. DNA ligase joins the newly synthesized segments. Incision activity is determined by 3 genes in E.coli . : uvrA,uvrB and uvrC (ultraviolet repair endoI ). Many human diseases may result from inability of exicision repair. E.g. xeroderma pigmentosum .

9. Xeroderma pigmentosum Autosomal recessive mutations in several complementation groups Extreme sensitivity to sunlight Predisposition to skin cancer (mean age of skin cancer = 8 yrs vs. 60 for normal population)

10. THYMINE DIMER Effect of thymine dimer on Dna replication: Pol III reaches a thymine dimer , the rep. fork fails to advance. But thymine dimer capable of forming hydrogen bond with two adenines. Dimer introduces distortion into the helix. Editing function removes the adenine. The cycle begins again an adenine is added and then it is removed. The net result is that the polymerase is stalled at the site of the dimer . (ultraviolet light induced idling process).

11. THYMINE DIMER There are 2 diff. ways in which DNA synthesis can get going again: 1. Post dimer initiation ( Recombinational repair) 2. Trans dimer synthesis (SOS repair)

12. RECOMBINATIONAL REPAIR One way to deal with a thymine dimer block is pass it by and initiate chain growth beyond the block. Post dimer initiation occurs after a pause of 5 sec. per thymine dimer . (unprimed initiation) The daughter strands have large gaps, because of unexcised thymine dimers . There is no way to produce viable daughter cells by continued replication alone. A recombination mechanism called sister-strand exchange forms double stranded molecule. In sister strand exchange, a single stranded segment free of any defects is excisied from a good strand on the homologous DNA segment at the replication fork and inserted into the gap created by excision of a thymine dimer . The combined action of Pol.I and ligase joins the inserted piece to adjacent regions, thus filling the gap. The gap formed in the donor mole. By excision is also filled by pol.I and ligase . Since it occurs after replication, it is called as postreplicational repair.

13. SOS REPAIR It is a bypass system, error-prone process, forms DNA strands but they are defective. It gives relaxation to editing system and allows polymerization to proceed across dimer ( tramsdimer synthesis) despite the distortion of the helix. New strands have a higher than normal no. of mispaired bases. Pol. III is modified in such a way, so it can continue chain growth without being stalled at damaged sites. The progeny will be mutants. It is cause of uv -induced mutagenesis.