Poly(ADPR)polymerase 1 and 2 (PARP-1, PARP-2) are nuclear enzymes which function is based on specific interactions with DNA and nuclear proteins. PARPs targets include proteins involved in DNA replication, repair, and transcription and their function can be modulated either by protein-protein interaction with native PARP-1 and by non-covalent interaction with poly(ADP-ribose) (pADPR) linked to the auto-modified PARP-1. Moreover, the association of pADPR and PARP-1 with the nuclear matrix (NM) has been reported, based on the poly(ADP-ribosyl)ation of nuclear matrix proteins (NMPs). In the present article, by the use of DNA and protein cross-linking reactions, by cis-diamminedichloroplatinum II (cDDP) and sodium tetrathionate (NaTT) respectively, we present more evidences about the association of PARP-1, PARP-2, and PARPs related proteins with the NM. Our findings confirmed that NM could be seen as a fraction greatly enriched in transcription factors (i.e., C/EBP-beta) and enzymes (DNA Topo II, DNA PK) that co-localize with PARP-1 and -2 at the matrix associated regions (MARs) of chromatin. Moreover, pADPR contributes to PARP-1 localization at the NM, showing that PARP(s) activity co-operates to the functions of this nuclear fraction.
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.
Poly(ADPR)polymerase (PARP) is a chromatin-associated enzyme with a presumptive role in DNA repair during replication and recovery from strand breaks caused by genotoxic agents. It catalyses the attachment and elongation of ADPribose polymers (pADPR) to a variety of acceptor proteins (including PARP itself, and histones) and is particularly active in the testis where its expression varies according to the stage of germ cell differentiation. PARP degradation is also one of the classic indicators of apoptosis. In this investigation we have examined the effects of heat stress on the adult rat testis with respect to the concentration and activity of PARP, the nature of the pADRP nuclear acceptor proteins, the length of ADPR polymers and the activity of the ADPR depolymerizing enzyme, poly(ADPR)glycohydrolase (PARG). Our results show a significant reduction in the concentration and activity of PARP 4 and 8 days after artificial cryptorchidism, but no significant changes were observed in PARG activity or in pADPR length. Unexpectedly, the apoptotic degradation of PARP was not detected following heat stress. These results confirm that PARP gene expression is developmentally regulated during spermatogenesis and indicate that it is suppressed coincidentally with the loss of meiotic spermatocytes during artificial cryptorchidism.
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