Poly(ADP-ribose) polymerase-1 dimerizes at a 5' recessed DNA end in vitro: a fluorescence study.
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 probably via its contribution to DNA base excision repair. In this study, we investigated the association of the amino-terminal DNA binding domain of human PARP-1 (hPARP-1 DBD) with a 5' recessed oligonucleotide mimicking a telomeric DNA end. We used the fluorescence of the Trp residues naturally occurring in the zinc finger domain of hPARP-1 DBD. Fluorescence intensity and fluorescence anisotropy measurements consistently show that the binding stoichiometry is two proteins per DNA molecule. hPARP-1 was found to bind the 5' recessed DNA end with a binding constant of approximately 10(14) M(-2) if a cooperative binding model is assumed. These results indicate that hPARP-1 DBD dimerizes during binding to the DNA target site. A footprint experiment shows that hPARP-1 DBD is asymmetrically positioned at the junction between the double-stranded and the single-stranded telomeric repeat. The largest contribution to the stability of the complex is given by nonionic interactions. Moreover, time-resolved fluorescence measurements are in line with the involvement of one Trp residue in the stacking interaction with DNA bases. Taken together, our data open new perspectives for interpretation of the selective binding of hPARP-1 to the junction between double- and single-stranded DNA.
UMR 7034 du CNRS, Laboratoire de Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, Faculté de Pharmacie, Université Louis Pasteur, 74, Route du Rhin, F-67401 Illkirch Cedex, France.
Emmanuelle
E
Pion
Elisa
E
Bombarda
Patrick
P
Stiegler
G Matthias
GM
Ullmann
Yves
Y
Mély
Gilbert
G
de Murcia
Dominique
D
Gérard
D000595:Amino Acid Sequence; D004247:DNA / Q000737:chemistry*; D019281:Dimerization; D008969:Molecular Sequence Data; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
D004247:DNA; D011065:Poly(ADP-ribose) Polymerases