title: Structural and biophysical studies of human PARP-1 in complex with damaged DNA.
abstract: The enzyme poly(ADP-ribose) polymerase-1 (PARP-1) is a global monitor of chromatin structure and DNA damage repair. PARP-1 binds to nucleosomes and poly(ADP-ribosylates) histones and several chromatin-associated factors to expose specific DNA sequences to the cellular machinery involved in gene transcription and/or DNA damage repair. While these processes are critical to genomic stability, the molecular mechanisms of how DNA damage induces PARP-1 activation are poorly understood. We have used biochemical and thermodynamic measurements in conjunction with small-angle X-ray scattering to determine the stoichiometry, affinity, and overall structure of a human PARP-1 construct containing the entire DNA binding region, the zinc ribbon domain, and automodification domains (residues 1-486). The interaction of this PARP-1 protein construct with three different DNA damage models (DNA constructs containing a nick, a blunt end, or a 3' extension) was evaluated. Our data indicate that PARP-1 binds each DNA damage model as a monomer and with similar affinity, in all cases resulting in robust activation of the catalytic domain. Using small-angle X-ray scattering, we determined that the N-terminal half of PARP-1 behaves as an extended and flexible arrangement of individually folded domains in the absence of DNA. Upon binding DNA, PARP-1 undergoes a conformational change in the area surrounding the zinc ribbon domain. These data support a model in which PARP-1, upon binding DNA, undergoes a conformational change to become an active nuclear enzyme.
authors: Department of Biochemistry and Molecular Biology, Colorado State University, Howard Hughes Medical Institute, Fort Collins, CO 80523, USA.
authors: Wayne
authors: W
authors: Lilyestrom
authors: Mark J
authors: MJ
authors: van der Woerd
authors: Nicholas
authors: N
authors: Clark
authors: Karolin
authors: K
authors: Luger
mesh_terms: D001483:Base Sequence; D055592:Biophysical Phenomena; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D017931:DNA Primers / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D008027:Light; D046911:Macromolecular Substances; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D013816:Thermodynamics; D014961:X-Ray Diffraction
chemical_list: D017931:DNA Primers; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases