Mol. Hum. Reprod. Advance Access originally published online on August 31, 2007
Molecular Human Reproduction 2007 13(11):821-828; doi:10.1093/molehr/gam062
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Differential contribution of poly(ADP-ribose)polymerase-1 and -2 (PARP-1 and -2) to the poly(ADP-ribosyl)ation reaction in rat primary spermatocytes
Department of Structural and Functional Biology, University ‘Federico II’, Via Cinthia Monte S. Angelo, 80126 Naples, Italy
1 Correspondence address. Tel: +39-81-679165; Fax: +39-81-679233; E-mail: quesada{at}unina.it
Poly(ADP-ribose)polymerases (PARP-1 and -2) are activated by DNA strand breaks to synthesize protein-bound ADP-ribose polymers from NAD+. The two enzymes are overexpressed in rat spermatocytes and are likely to play a role in meiosis. Indeed parp-2–/– mice, but not parp-1 knockouts, show hypofertility. Aside, PARP-1 and PARP-2 are both involved in DNA damage repair and signalling, but their relative contributions to such processes remain as yet unknown, largely because of the lack of PARP isoform-specific inhibitors that has precluded in vivo studies. Here, we used permeabilized rat primary spermatocytes or isolated spermatocyte nuclei and radiolabelled NAD+ to investigate potential isoform-specific effects on basic features of the poly(ADP-ribosyl)ation reaction, including size of ADP-ribose polymers at different NAD+ concentrations, extent of auto- versus etheromodification, and modulation of such reactions by the PARP inhibitor, PJ34. We found that PARP-1 automodification prevailed over PARP-2 modification. In addition, over 50% of cellular poly(ADP-ribose) was covalently bound to histones H1 and H2. The inhibitory effect of PJ34 appeared to be targeted mainly to the elongation step of the reaction. We propose that a different propensity of PARP-1 and PARP-2 to undergo automodification and/or catalyze etheromodification, both in terms of number of enzyme molecules being involved and amount of bound poly(ADP-ribose), may underlie distinct roles in the regulation of spermatocyte functions.
Key words: PARP-1/PARP-2/histone H1/histone H2/rat primary spermatocytes
Submitted on July 25, 2007; accepted on August 21, 2007.