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poly(ADP-ribosyl)ation
Frederic R. Sallmann
Research Experience : Poly(ADP-ribosyl)ation (clonage of sPARP-1, DNA repair and Apoptosis)
 Poly(ADP-ribosyl)ation (pADPr) is a covalent post-translational modification of nuclear proteins that is activated by DNA strand interruptions. It has been reported to take place in many nuclear processes such as DNA repair, replication and transcription. Sofar, pADPr synthesis is known to be catalyzed by poly(ADP-ribose) polymerase-1 (PARP-1). This enzyme is a multifunctional protein that comprises three domains: an amino-terminal DNA binding domain; a carboxy-terminal catalytic domain; and an automodification domain. During apoptosis PARP-1 is cleaved by caspases generating thus two apoptotic fragments and thereby the inactivation of PARP-1.
 To study cellular role of PARP-1, many laboratories have developed mice knockout in PARP-1.
 Even though the enzyme PARP-1 is not found in those PARP-1 (-/-) cells, we evidenced a PARP activity independent of DNA strand breaks. We cloned the cDNA of the enzyme responsible for this activity, and called it sPARP-1. We characterized the enzyme, and demonstrated that sPARP-1 is a nuclear enzyme that synthesizes similar pADPr than PARP-1, and whose activity is independent of the presence of DNA strand breaks. We furthermore demonstrated that sPARP-1 is an alternative product of the PARP-1 gene, resulting of an alternative transcription start-point. See some figures. This work is in press for the Journal of Biological Chemistry.

 Cells and mice lacking PARP-1 are hypersensitives to genotoxic agents. This hypersensitivity could come from a defect in DNA repair. We therefore took these cells as a template to study the role of PARP-1 in DNA repair. We dissected the DNA base excision repair mechanism (BER) employing a biochemical assay elaborated and an in vivo assay. We demonstrated that the four steps of BER are normal in these cells. Thus the hypersensitivity of mice and cells lacking PARP-1 cannot come from a defect in BER. However, even if these results suggest that PARP-1 is not involved in BER, it is not proved. Indeed, many sub-pathways of BER have been evidenced these latest years, and one can suggest that one of those sub-pathways can take over. We proposed a mechanism highlighting a potential role of pADPr synthesized during BER.(see figures)

 We demonstrated that the apoptotic DBD of PARP-1 has the capacity to inhibit BER. We used a biochemical assay and an in vivo assay. We suggest that the cleavage of PARP-1during apoptosis can lead to inhibition of BER helping the cell to commit suicide. Our finding has been confirmed by atomic force microscopy experiments from another group.They showed that the apoptotic DBD of PARP-1 remained bound to DNA strand break, thus preventing access to other proteins such as PARP-1 or DNA repair enzymes. This feature is being explored to potentially set up new cancer therapy. The goal is to overexpress the apoptotic DBD of PARP-1 in cancer cells during radiotherapy treatment. This treatment leads to formation of DNA breaks; the overexpressed apoptotic DBD of PARP-1 will bind to them preventing their repair and thereby triggering apoptosis. This therapy might allow a decrease in the intensity of irradiation used and thereby avoid the development of secondary cancers.
Publications:

F.R. Sallmann, S. Bourassa, J. Saint-Cyr & G.G. Poirier. 1997. Analysis of caspase activation during apoptosis: utilization of antibodies specific for caspase site of Poly(ADP-ribose) polymerase. Biochem. Cell Biol. 75: 451-456

 F.R. Sallmann, Y.D. Plancke & G.G. Poirier. 1998. Rapid detection of Poly(ADP-ribose) polymerase by enzyme-linked immunosorbent assay during its purification and improvement of its purification. Mol. cell. Biochem. 185: 199-203

 E.B. Affar, P.J. Duriez, R.G. Shah, F.R. Sallmann, S. Bourassa, J-H Küpper, A. Bürkle & G.G. Poirier, 1998. Immuno-dot blot method for detection of poly(ADP-ribose) synthesized in vitro and in vivo. Anal. Biochem. 259: 280-283

F.R. Sallmann, M.D. Vodenicharov, Z-Q. Wang and G.G.Poirier. Characterization of sPARP-1: an alternative product of PARP-1 gene with poly(ADP-ribose) polymerase activity independent of DNA strand breaks J. Biol. Chem. Vol. 275: 15504-15511

 M.D. Vodenicharov, F.R. Sallmann, Z-Q. Wang, M.S. Satoh and G.G. Poirier. Base excision repair in cells lacking poly(ADP-ribose) polymerase-1 (PARP-1). (to be submitted to Biochemistry.)

D. d'Amours, F.R. Sallmann, V.M. Dixit and G.G. Poirier. Poly(ADP-ribose) polymerase cleavage by caspase-3: transdominant inhibition of enzyme activity and of base excision repair. (for Exp. Cell Res.)

F.R. Sallmann and G.G. Poirier. May 11, 1998. Analyse moléculaire du rôle de la poly(ADP-ribose) polymérase dans l'apoptose. 66e Congrès de l'Acfas. Université Laval, Québec, Qc, Canada.

F.R. Sallmann, S. Bourassa, J. Saint-Cyr & G.G. Poirier. February 1997. Analysis of caspase activation during apoptosis: utilization of antibodies specific for caspase site of Poly(ADP-ribose) polymerase. 7th Annual Winternational Symposium; Apoptosis and DNA repair. Québec, Qc, Canada

F.R. Sallmann, S. Bourassa, J. Saint-Cyr & G.G. Poirier. June 1997. Characterization of antipeptide antibodies specific for the cleavage site of PARP by ICE-like protease. 40th annual meeting of Canadian Federation of Biological Societies. Québec, Qc, Canada

F.R.Sallmann, M.D. Vodenicharov, S. Gobeil, Masahiko S. Satoh, Z-Q. Wang and G.G.Poirier. 11-15 November 1998. Does the gene disruption of Poly(ADP-ribose) polymerase affect base excision repair process? Endogenous Sources of Mutations, an AACR special conference. Fort-Myers, Florida .
Links:


(English version will be available soon)

pADPr Group (picture)

 

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