Poly(ADP‐ribose) polymerase‐1 protects excessive DNA strand breaks from deterioration during repair in human cell extracts

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Abstract
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.