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Dna repair
- 1. DNA REPAIR Dr.Khushbu Soni 3rd Year Resident Dept. of Biochemistry
- 2. DNA damage, if not repaired, may affect replication and transcription, leading to mutation or cell death DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. What is DNA repair?
- 3. Efficient DNA replication DNA replication is highly efficient process Replication error rate is only 1 defect in 10 8 nucleotides DNA repair rate is 99% Unrepaired error rate is only 1 in 10 10 nucleotides Human genome=6.4x10 9 base pairs
- 4. DNA damage Activation of cell cycle check points DNA repair SOS Response (synthesis of repair enzymes Trans lesion synthesis/ bypas-Photoreactivation -Demethylation MMR BER NER HR NHEJ FAILURE Senescence Apoptosis Mutation If excess damage If excess damage No effect Cancer Death
- 5. TYPE OF DAMAGE DAMAGE MECH. REPAIR MECH. Single Base Alteration Depurination BER Deamination BER Alkylation BER Base Analogue Incorporation BER Mismatch Base MMR Double Base Alteration Pyrimidine dimer NER Purine dimer NER Chain Break Single stranded break HR,NHEJ Double stranded break HR,NHEJ Cross linking Between DNA-DNA HR Between DNA-Protein ? Polymerase slippage Replication error in microsettelite MMR & NER
- 6. Cell cycle check point
- 7. DNA damage Activation of cell cycle check points DNA repair SOS Response (synthesis of repair enzymes Trans lesion synthesis/ bypas-Photoreactivation -Demethylation MMR BER NER HR NHEJ FAILURE Senescence Apoptosis Mutation If excess damage If excess damage No effect Cancer Death
- 8. SOS response SOS repair occurs when cells are overwhelmed by UV damage - this allows the cell to survive but at the cost of mutagenesis. SOS response only triggered when other repair systems are overwhelmed by amount of damage so that unrepaired DNA accumulates in the cell.
- 11. DNA damage Activation of cell cycle check points DNA repair SOS Response (synthesis of repair enzymes Trans lesion synthesis/ bypas-Photoreactivation -Demethylation MMR BER NER HR NHEJ FAILURE Senescence Apoptosis Mutation If excess damage If excess damage No effect Cancer Death
- 12. Direct reversal of DNA damage Photoreactivation (the enzyme DNA photolyase captures energy from light ) It is found in plants and some prokaryotes
- 13. Methylation of DNA by alkylating agents
- 14. Direct Repair: Reversal of O6 methyl G to G by methyltransferase
- 15. DNA damage Activation of cell cycle check points DNA repair SOS Response (synthesis of repair enzymes Trans lesion synthesis/ bypas-Photoreactivation -Demethylation MMR BER NER HR NHEJ FAILURE Senescence Apoptosis Mutation If excess damage If excess damage No effect Cancer Death
- 16. Mismatch repair(MMR) • MMR system is an excision/resynthesis system that can be divided into 4 phases: • (i) recognition of a mismatch by MutS proteins • (ii) recruitment of repair enzymes • (iii) excision of the incorrect sequence, • (iv) resynthesis by DNA polymerase using the parental strand as a template.
- 17. When MMR is implicated? When a mismatch base is encountered When DNA polymerase slippage forms loop type defect During replication,
- 18. In prokaryotes like E.coli… Mut proteins Mut S Mut L Mut H Scans DNA and recognize the mismatch base on daughter strand -Links Mut S and Mut H -Activates Mut H -Binds the complex to hemi methylated GATC sequence -Helicase activity
- 20. Clinical importance hereditary nonpolyposis colon cancer ( HNPCC ) Faulty mismatch repair Mutation in gene hMSH2 and hMLH1 Defective mismatch repair mechanism Autosomal dominant carcinoma
- 22. In eukaryotes- MutS homologs: Msh2/Msh6 (MutSα) Msh2/Msh3 (MutSβ) MutL homologs: MLH1 and PMS2(MutLα) MLH1 and PMS1(MutLβ) MLH1 and MLH3(MutLγ)
- 23. TYPE OF DAMAGE DAMAGE MECH. REPAIR MECH. Single Base Alteration Depurination BER Deamination BER Alkylation BER Base Analogue Incorporation BER Mismatch Base MMR Double Base Alteration Pyrimidine dimer NER Purine dimer NER Chain Break Single stranded break HR,NHEJ Double stranded break HR,NHEJ Cross linking Between DNA-DNA HR Between DNA-Protein ? Polymerase slippage Replication error in microsettelite MMR & NER
- 24. Microsatellite instability (MSI) It is a condition manifested by damaged DNA due to defects in the normal DNA repair process. Microsatellites are repeated sequences of DNA A dinucleotide repeat of CA, is most common DNA polymerase slips out from these sequences while replication and forms loop This is corrected by MMR and NER mechanism
- 25. Defect in MMR will result in increase or decrease length Microsatellites It will cause DNA mutation
- 27. TYPE OF DAMAGE DAMAGE MECH. REPAIR MECH. Single Base Alteration Depurination BER Deamination BER Alkylation BER Base Analogue Incorporation BER Mismatch Base MMR Double Base Alteration Pyrimidine dimer NER Purine dimer NER Chain Break Single stranded break HR,NHEJ Double stranded break HR,NHEJ Cross linking Between DNA-DNA HR Between DNA-Protein ? Polymerase slippage Replication error in microsettelite MMR & NER
- 28. Base excision repair Variety of DNA glycosylases, for different types of damaged bases. They scan the genome and flips out the wrong base AP endonuclease recognizes sites with a missing base; cleaves sugar-phosphate backbone. (Deoxyribose phosphate lyase) removes the sugar- phosphate lacking the base.
- 30. Nucleotide Excision Repair Used for repair of DNA adducts EXAMPLES: thymine-thymine dimers Produced by chemical and UV radiation damage
- 31. Nucleotide Excision repair in E.coli 1.UvrA and UvrB scan DNA to identify a distortion 2. UvrA leaves the complex ,and UvrB melts DNA locally around 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
- 33. Nucleotide excision repair in eukaryotes Two major pathways Global genome repair Transcription coupled repair
- 34. Type of protein Function DDB1 ,DDB2(XPE) XPC Recognize damage XPG -- Stabilizes TFIIH -- Endonuclease activity XPD (subunit of TFIIH) Act as helicase XPB (subunit of TFIIH) Act as ATPase XPA Involved in damage verification XPF endonuclease
- 36. Clinical Importance Xeroderma pigmentosum is an autosomal recessive genetic disease . The clinical syndrome include marked sensitivity to sunlight ( UV rays ) with subsequent formation of multiple skin cancers & premature death . The risk of developing skin cancer is increased 1000 to 2000 fold. Cells cultured from patients with xeroderma pigmentosum exhibit low activity for the nucleotide excision repair process
- 37. TYPE OF DAMAGE DAMAGE MECH. REPAIR MECH. Single Base Alteration Depurination BER Deamination BER Alkylation BER Base Analogue Incorporation BER Mismatch Base MMR Double Base Alteration Pyrimidine dimer NER Purine dimer NER Chain Break Single stranded break HR,NHEJ Double stranded break HR,NHEJ Cross linking Between DNA-DNA HR Between DNA-Protein ? Polymerase slippage Replication error in microsettelite MMR & NER
- 38. Double-strand break repair NO TEMPLATE FOR REPAIR!!
- 39. End-joining repair of nonhomologous DNA
- 40. Two proteins are involved in the non homologous rejoining of a DNA break . Ku protein, a hetero dimer with two subunits bind to free DNA ends & has latent ATP dependent helicase activity . The DNA bound Ku hetero dimer recruits an unusual DNA dependent Protein kinase ( DNA – PK )
- 41. DNA – PK has a binding site for DNA free ends It allows the approximation of the 2 separated ends . The free end DNA/Ku/DNA – PK complex activates the kinase activity in the later. DNA – PK reciprocally phosphorylates Ku .
- 42. DNA – PK then dissociates from the DNA & Ku, resulting in activation of the Ku helicase. This results in unwinding of the 2 ends. The unwound approximated DNA forms base pairs. The extra nucleotide tails are removed by an exonuclease & the gaps are filled and closed by DNA ligase .
- 43. Homologous recombination Double strand break MRN complex binds to DNA on either side of the break Various proteins will trim back 5’ ends Production of 3’ overhangs on ssDNA RPA will cover ssDNA & prevents its own winding Rad 51 produce nucleoprotein on ss DNA
- 44. Strand invasion in identical duplex D-loop formation between 3’ overhang and homologous chromosome DNA polymerase will extend it Holiday junction formation DSBR pathway SDSA pathway Cross over product Non cross over product
- 48. DNA damage Activation of cell cycle check points DNA repair SOS Response (synthesis of repair enzymes Trans lesion synthesis/ bypas-Photoreactivation -Demethylation MMR BER NER HR NHEJ FAILURE Senescence Apoptosis Mutation If excess damage If excess damage No effect Cancer Death
- 49. Trans lesion DNA synthesis Occurs when the above repairs are not efficient enough Prevents cell from having un-replicated chromosome at the cost of some point mutation enables replication to proceed across DNA damage