ASSOCIATION OF POLY(ADP-RIBOSE) POLYMERASE WITH NUCLEAR SUBFRACTIONS CATALYZED WITH SODIUM TETRATHIONATE AND HYDROGEN-PEROXIDE CROSS-LINKS

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme which catalyzes the transfer of ADP-ribose units from NAD(+) to a variety of nuclear proteins under the stimulation of DNA strand break. To examine its rol e in DNA repair, we have been studying the interaction of PARP with ot her nuclear proteins using disulfide cross-linking, initiated by sodiu m tetrathionate (NaTT). Chinese Hamster Ovary (CHO) cells were extract ed sequentially with Nonidet P40 (detergent), nucleases (DNase + RNase ), and high salt (1.6 M NaCl) with and without the addition of a sulfh ydryl reducing agent. The residual structures are referred to as the n uclear matrix, and are implicated in the organization of DNA repair an d replication. Treatment of the cells with NaTT causes the crosslinkin g of PARP to the nuclear matrix. Activating PARP by pretreating the ce lls with H2O2 did not increase the cross-linking of PARP with the nucl ear matrix, suggesting a lack of additional interaction of the enzyme with the nuclear matrix during DNA repair. Both NaTT and H2O2 induced crosslinks of PARP that were extractable with high salt. To shorten th e procedure, these crosslinks were extracted from cells without nuclea ses and high salt treatment, using phosphate buffer. Using western blo tting, these crosslinks appeared as a smear of high molecular weight s pecies including a possible dimer of PARP at 230 kDa, which return to 116 kDa following reduction with beta-mercaptoethanol.