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

2. Andrew J. Pierce TOX 780 Single-strand Nicks Any covalent disruption in the phosphodiester backbone in ONE of the two DNA strands in double-helical DNA Ligatable: needs a 5'-PO 4 end and a 3'-OH end Otherwise, need some end processing AP-endonuclease, polynucleotide kinase, DNA-polymerase  Reactive oxygen species Base hydrolysis Base excision repair 1.5 x 10 5 per cell per day

3. Andrew J. Pierce TOX 780 Pol  Can cleave to the 3' side of abasic sites Has dRP-lyase activity APE1 (AP endonuclease-1) Can cleave to the 5' side of abasic sites and damaged termini to leave a 3' OH Base Excision Repair Marti TM, Fleck O. DNA repair nucleases Cell Mol Life Sci. 2004 Feb;61(3):336-54

4. Andrew J. Pierce TOX 780 XRCC1 Interactions

5. Andrew J. Pierce TOX 780 XRCC1 (N-term) DNA pol  Nicked DNA XRCC1 Holds It Together

6. Andrew J. Pierce TOX 780 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  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  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. Andrew J. Pierce TOX 780 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. Andrew J. Pierce TOX 780 Ku 70 Ku 80 Ku 70 Ku 80 DNA PK CS Ku 70 Ku 80 Lig IV XRCC4 DNA PK CS Ku 70 Ku 80 Lig IV XRCC4 DNA PK CS Ku 70 Ku 80 Ku 80 Ku 70 DNA PK CS Mammalian End-joining Proteins Protein Missing Ku 70/80 DNA-PK CS XRCC4 / DNA ligase IV V(D)J Rec. no Coding Joints no Signal Joints no yes* no

9. Andrew J. Pierce TOX 780 Ku70/80 Heterodimer Binds DNA Double-strand Ends In Preparation for End-joining

10. Andrew J. Pierce TOX 780 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)