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Gain-of-function of poly(ADP-ribose) polymerase-1 upon cleavage by apoptotic proteases: implications for apoptosis

¹ Wellcome/CRC Institute, Tennis Court Road, Cambridge, CB2 1QR, UK
² Department of Biology, MIT 77, Massachusetts Avenue, Cambridge, MA, USA
³ Department of Molecular Oncology, Genentech Inc., 460 Point San Bruno Blvd, South San Francisco, CA 94080, USA
⁴ Unit of Health and Environment, Laval University Medical Research Center, CHUQ, Faculty of Medicine, Laval University, Québec, Canada

*Author for correspondence (e-mail: guy.poirier{at}crchul.ulaval.ca)

Accepted July 6, 2001

Poly(ADP-ribosyl)ation is an important mechanism for the maintenance of genomic integrity in response to DNA damage. The enzyme responsible for poly(ADP-ribose) synthesis, poly(ADP-ribose) polymerase 1 (PARP-1), has been implicated in two distinct modes of cell death induced by DNA damage, namely apoptosis and necrosis. During the execution phase of apoptosis, PARP-1 is specifically proteolyzed by caspases to produce an N-terminal DNA-binding domain (DBD) and a C-terminal catalytic fragment. The functional consequence of this proteolytic event is not known. However, it has recently been shown that overactivation of full-length PARP-1 can result in energy depletion and necrosis in dying cells. Here, we investigate the molecular basis for the differential involvement of PARP-1 in these two types of cellular demise. We show that the C-terminal apoptotic fragment of PARP-1 loses its DNA-dependent catalytic activity upon cleavage with caspase 3. However, the N-terminal apoptotic fragment, retains a strong DNA-binding activity and totally inhibits the catalytic activity of uncleaved PARP-1. This dominant-negative behavior was confirmed and extended in cellular extracts where DNA repair was completely inhibited by nanomolar concentrations of the N-terminal fragment. Furthermore, overexpression of the apoptotic DBD in mouse fibroblast inhibits endogenous PARP-1 activity very efficiently in vivo, thereby confirming our biochemical observations. Taken together, these experiments indicate that the apoptotic DBD of PARP-1 acts cooperatively with the proteolytic inactivation of the enzyme to trans-inhibit NAD hydrolysis and to maintain the energy levels of the cell. These results are consistent with a model in which cleavage of PARP-1 promotes apoptosis by preventing DNA repair-induced survival and by blocking energy depletion-induced necrosis.

Key words: PARP-1, Transdominant, Apoptosis, Necrosis, DNA-binding domain

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