Association of poly(ADP-ribose) polymerase with nuclear subfractions catalyzed with sodium tetrathionate and hydrogene peroxide crosslinks |
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Authors: | Serge Desnoyers James B. Kirkland Guy G. Poirier |
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Affiliation: | (1) Research Group on Poly(ADP-ribose) Metabolism, Health and Environment Unit, CHUL Research Center and Laval University, GIV 4G2 Sainte-Foy, Quebec, Canada;(2) Present address: Cold Spring Harbor Laboratory, 1 Bungtown road, 11724 Cold Spring Harbor, NY, USA;(3) Present address: Department of Nutritional Sciences, University of Guelph, NIG 2W1 Ontario, Canada |
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Abstract: | 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 role in DNA repair, we have been studying the interaction of PARP with other nuclear proteins using disulfide cross-linking, initiated by sodium tetrathionate (NaTT). Chinese Hamster Ovary (CHO) cells were extracted sequentially with Nonidet P40 (detergent), nucleases (DNase + RNase), and high salt (1.6 M NaCl) with and without the addition of a sulfhydryl reducing agent. The residual structures are referred to as the nuclear matrix, and are implicated in the organization of DNA repair and replication. Treatment of the cells with NaTT causes the crosslinking of PARP to the nuclear matrix. Activating PARP by pretreating the cells with H2O2 did not increase the cross-linking of PARP with the nuclear 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 the procedure, these crosslinks were extracted from cells without nucleases and high salt treatment, using phosphate buffer. Using western blotting, these crosslinks appeared as a smear of high molecular weight species including a possible dimer of PARP at 230 kDa, which return to 116 kDa following reduction with -mercaptoethanol. |
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Keywords: | crosslinking sodium tetrathionate protein-protein interaction poly(ADP-ribose) polymerase oxidative stress |
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