Poly(ADP-ribose) polymerase-1 protects excessive DNA strand breaks from deterioration during repair in human cell extracts.
Base excision repair (BER), a major pathway for the removal of simple lesions in DNA, requires the co-ordinated action of several repair and ancillary proteins, the impairment of which can lead to genetic instability. We here address the role of poly(ADP-ribose) polymerase-1 (PARP-1) in BER. Using an in vitro cross-linking assay, we reveal that PARP-1 is always involved in repair of a uracil-containing oligonucleotide and that it binds to the damaged DNA during the early stages of repair. Inhibition of PARP-1 poly(ADP-ribosyl)ation by 3-aminobenzamide blocks dissociation of PARP-1 from damaged DNA and prevents further repair. We find that excessive poly(ADP-ribosyl)ation occurs when repair intermediates containing single-strand breaks are in excess of the repair capacity of the cell extract, suggesting that repeated binding of PARP-1 to the nicked DNA occurs. We also find increased sensitivity of repair intermediates to nuclease cleavage in PARP-deficient mouse fibroblasts and after depletion of PARP-1 from HeLa whole cell extracts. Our data support the model in which PARP-1 binding to DNA single-strand breaks or repair intermediates plays a protective role when repair is limited.
Parsons
Jason L
JL
MRC Radiation and Genome Stability Unit, Harwell, Oxfordshire, UK.
Dianova
Irina I
II
Allinson
Sarah L
SL
Dianov
Grigory L
GL
D000818:Animals; D001483:Base Sequence; D002457:Cell Extracts / Q000737:chemistry* / Q000235:genetics; D002460:Cell Line; D003432:Cross-Linking Reagents; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008954:Models, Biological; D000071137:Poly (ADP-Ribose) Polymerase-1; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000172:deficiency / Q000378:metabolism*; D011485:Protein Binding; D000076105:X-ray Repair Cross Complementing Protein 1
D002457:Cell Extracts; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D000076105:X-ray Repair Cross Complementing Protein 1; D004247:DNA; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D019951:DNA Polymerase beta