Abstract

The presence of damaged DNA in the cell activates signal transduction pathways. These pathways trigger cell cycle arrest and repair mechanisms ultimately leading to programmed cell death or to cell survival. Activation of poly (ADP-ribose) polymerase 1 (PARP-1) is an immediate cellular reaction to DNA strand breakage as induced by alkylating agents, ionizing radiation or oxidants. The resulting formation of protein-bound poly (ADP-ribose) facilitates survival of proliferating cells under conditions of DNA damage owing to chromatin structure opening and/or recruitment of DNA repair enzymes and factors. Evidence has emerged that this posttranslational modification is involved in the maintenance of genomic stability. In the present study fluorescence spectroscopy and gel shift assay were used to investigate the DNA binding properties of the wild-type (wt) zinc finger domain of hPARP-1 (DBD) and a derivative (W51A) mutated in the first zinc-finger. Double-stranded oligonucleotides mimicking an open telomeric DNA-end were used as probes. Fluorimetric titrations unambiguously showed that the binding stoechiometry was two wt hPARP-1 DBD/DNA probe. Moreover, time resolved fluorescence measurements revealed that at least one of the Trp residues is involved in stacking interaction with the bases. In contrast, no interaction was detected with W51A. To understand the lack of interaction with the (W51A) mutant, circular dichroism experiments were performed showing a large conformational change. Therefore, Trp 51 seems to be essential to the proper protein conformation in order to allow PARP-1/DNA interaction.References: 1.Zhou BB, Elledge SJ: Nature, 2000; 408: 433-439 2.De Murcia G, Shall S: From DNA damage and stress signaling to cell death. Poly ADP-ribosylation reactions. Oxford University Press, Oxford 2000, UK 3.D’amours D, Desnoyers S, D’Silva I, Poirier GG: Biochem J, 1999; 342(Pt 2): 249-268 4.Simbulan-Rosenthal CM, Haddad BR, Rosenthal DS et al: Biochimie, 1999; 81: 69-75 5.Bombarda E, Ababou A, Vuilleumier C et al: Biophys J, 1999; 76: 1561-70 6.Pion E, Bombarda E, Stiegler P et al: 2003; submitted

Keywords: DNA binding, repair, cancer, fluorescence spectroscopy