Mechanism of repair

Student at Alagappa University, Alagappa Nagar, Karaikudi
8. Dec 2016

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Mechanism of repair

  1. Molecular biology Mechanism of repair B. C. Muthubharathi I M.Sc., Biotechnology
  2. DNA Damage – A short note  Both physical and chemical agents have the ability to alter the primary structure of DNA  DNA susceptible to environmental damages – Exogenous, Endogenous  Ionizing radiation  Exposure to chemicals  UV rays  Cell’s metabolic products  Absorption of thermal energy during metabolism – Tautomeric shift (rearrangement of bases)  Leads to alteration, Mutation etc.,
  3. Tautomeric shift  Rearrangement of basepairs  occur during normal metabolism  Results in the production of structural isomers of a base  Enhance the mispairing
  4. But the system is lucky  Repair mechanism 1. Automatic 2. Proteins and enzymes are available  Process I. Excision / removal of damaged section II. Reconstruction of complementary removed section
  5. Types of repair mechanism  Excision repair mechanism a. Nucleotide excision repair b. Base excision repair c. Mismatch excision repair  Photo reactivation  Recombinational repair  SOS response  Adaptive response  Heat shock repair
  6. Nucleotide excision repair  Cut – patch mechanism  Bulky lesions like thymine dimers can be removed and rearranged  Steps 1. Recognition of bulky lesion in DNA 2. Hydrolyzing the phosphodiester bond in deoxyribose backbone in 5’ side of lesion 3. Excising the lesion ( along with a limited number of nucleotide on its 3’ end) 4. Filling the resultant gap with information given by complementary strand 5. Closing the nicked DNA to generate intact strand
  7. Pathways  Transcription coupled pathway template strand is repaired occur during transcription lesion is signaled by RNA polymerase  Global genomic pathway less efficient remaining of DNA is repaired much slow
  8. (1) damage recognition in the global pathway is mediated by an XPC-containing protein complex, whereas damage recognition in the transcription-coupled pathway is thought to be mediated by a stalled RNA polymerase in conjunction with a CSB protein (2) DNA strand separation (by XPB and XPD proteins, two helicase subunits of TFIIH (3) incision (by XPG on the 3’ side and the XPF–ERCC1 complex on the 5’ side) (4) excision, (5) DNA repair synthesis (by DNA polymerase ) (6) ligation (by DNA ligase I).
  9. NER in E. Coli  ABC excinuclease ( Uvr A, Uvr B, Uvr C)  A complex A2B ( Uvr A, Uvr B) which scans the DNA for any damage and binds to the lesion  Uvr A is dissociated and leaving Uvr B – DNA complex  Uvr C binds to Uvr B  12-13 nucleotides are removed by Uvr D helicase  The gap is filled by DNA polymerase I and DNA ligase
  10. Base Excision Repair  DNA glycosylase recognize DNA lesion and remove the N-glycosyl bond  This cleavage creates apurine or apyrimidine site (AP site or abasic site)  Several DNA glycosylase are found and each specific for a particular type of DNA lesions  DNA lesions may because of uracil formation( deamination of cytosine) 8-oxoguanine 3-methyladenine (transfer of methyl group)
  11. Example for BER  REPAIR OF 8-OXOGUANINE BY DNA GLYCOSYLASE • Inspection of G-C pair on the damaged DNA by DNA glycosylase • When the enzyme come across the oxoGC, it inserts a specific aminoacid chain to the DNA which creates a 180 of rotation. • Then the base fits to the active site of enzyme and cleaved form associated sugar • Once the altered purine or pyrimidine is removed the deoxyribose phosphate is remaining in the site and it is excised by AP endnuclease • Polymerization by DNA polymerase  • Final sealing is done by DNA Ligase III
  12. Mismatch repair  Mismatched basepair causes a distortion in the geometry double helix  Also called Methyl directed post replication repair system  The mismatches are nearly always corrected to reflect the information in the old (template) strand, so the repair system must somehow discriminate between the template and the newly syrrthesized strand  Steps • Tagging the template DNA with methyl group to differentiate from newly synthesized DNA • Methylation is done in N6- position of adenine in 5’ GATC sequence
  13. • The newly synthesized strand is repaired according to the information stored in the methylated parent strand • Mismatch repair system efficiently repair nearly 1000bp in hemi methylated strand • MutL and MutS proteins form a complex and binds to the mismatched basepair • MutH protein binds to the MutL. MutH has a site specific endonuclease activity Proteins involved MutL, MutS, MutH DNA helicase II exonuclease VII/ Rec J nuclease exonuclease I / exonuclease X
  14. Photoreactivation  DNA damages like pyrimidine dimer formation due to UV radiation  DNA photolyase enzymes are involved  The enzyme use energy utilized from absorbed light to reverse the damage  Photolyase has 2 cofactors – MTHF PolyGlu (N5,N10 –methenyltetrahryodfolylpolygultamate FADH