Single and Double-strand Break Repair - PowerPoint PPT Presentation

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Single and Double-strand Break Repair

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  1. Single and Double-strand Break Repair Andrew Pierce Microbiology, Immunology and Molecular Genetics Toxicology University of Kentucky TOX 780

  2. Single-strand Nicks Any covalent disruption in the phosphodiester backbone in ONE of the two DNA strands in double-helical DNA Reactive oxygen species Base hydrolysis Base excision repair 1.5 x 105 per cell per day Ligatable: needs a 5'-PO4 end and a 3'-OH end Otherwise, need some end processing AP-endonuclease, polynucleotide kinase, DNA-polymerase b

  3. Base Excision Repair APE1 (AP endonuclease-1) Can cleave to the 5' side of abasic sites and damaged termini to leave a 3' OH Polb Can cleave to the 3' side of abasic sites Has dRP-lyase activity Marti TM, Fleck O. DNA repair nucleases Cell Mol Life Sci. 2004 Feb;61(3):336-54

  4. XRCC1 Interactions

  5. XRCC1 Holds It Together DNA pol b XRCC1 (N-term) Nicked DNA

  6. Single-strand Nick Repair Summary The single-strand nick lesion is recognized by PARP which recruits XRCC1 or by XRCC1 directly XRCC1 forms a protein scaffold and recruits APE (AP-endonuclease) and PNK (polynucleotide kinase) to trim the DNA ends XRCC1 recruits DNA pol b for additional trimming and nucleotide replacement XRCC1 recruits DNA ligase III to restore covalent continuity to the repaired DNA strand Mouse knockouts of any of XRCC1, APE, DNA pol b are embryonic lethal XRCC1-deficient cell lines show 10x elevation in levels of sister chromatid exchange [conversion of single-strand nicks to double-stranded breaks]

  7. Natural Damage Ionizing radiation Reactive oxygen species Free radicals Cancer Treatment Chemotherapy Radiotherapy Programmed Breaks Rag 1/2 Spo11 Enzymatic Failure Topoisomerases Replication forks Double-strand Breaks Challenge Genome Stability

  8. Protein Missing Ku 70/80 DNA-PKCS XRCC4 / DNA ligase IV V(D)J Rec. no no no Coding Joints no no no Signal Joints no yes* no Mammalian End-joining Proteins Ku 80 Ku 70 DNA PKCS Ku 80 Ku 70 DNA PKCS Ku 80 Ku 70 Ku 80 Ku 70 DNA PKCS XRCC4 Ku 80 Ku 70 Lig IV Lig IV Ku 70 Ku 80 DNA PKCS XRCC4

  9. Ku70/80 Heterodimer Binds DNA Double-strand Ends In Preparation for End-joining

  10. Other Proteins That May Play an End-Processing Role Artemis: opens hairpins XPG (Fen1) (XRCC5): flap endonuclease, 5' terminated single-stranded flaps ERCC1/XPF (ERCC4): flap endonuclease, 3' terminated single-stranded flaps Fen-1 (P. furiosus)