title: Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro.
abstract: The existing methodologies for studying robust responses of poly (ADP-ribose) polymerase-1 (PARP-1) to DNA damage with strand breaks are often not suitable for examining its subtle responses to altered DNA without strand breaks, such as UV-damaged DNA. Here we describe two novel assays with which we characterized the interaction of PARP-1 with UV-damaged DNA in vivo and in vitro. Using an in situ fractionation technique to selectively remove free PARP-1 while retaining the DNA-bound PARP-1, we demonstrate a direct recruitment of the endogenous or exogenous PARP-1 to the UV-lesion site in vivo after local irradiation. In addition, using the model oligonucleotides with single UV lesion surrounded by multiple restriction enzyme sites, we demonstrate in vitro that DDB2 and PARP-1 can simultaneously bind to UV-damaged DNA and that PARP-1 casts a bilateral asymmetric footprint from -12 to +9 nucleotides on either side of the UV-lesion. These techniques will permit characterization of different roles of PARP-1 in the repair of UV-damaged DNA and also allow the study of normal housekeeping roles of PARP-1 with undamaged DNA.
authors.affiliation: Laboratory for Skin Cancer Research, CHU-Q (CHUL) Quebec University Hospital Research Centre &Laval University, 2705, Laurier Boulevard, Québec (QC) Canada G1V 4G2.
authors.forename: Nupur K
authors.initials: NK
authors.lastname: Purohit
authors.affiliation: Laboratory for Skin Cancer Research, CHU-Q (CHUL) Quebec University Hospital Research Centre &Laval University, 2705, Laurier Boulevard, Québec (QC) Canada G1V 4G2.
authors.forename: Mihaela
authors.initials: M
authors.lastname: Robu
authors.affiliation: Laboratory for Skin Cancer Research, CHU-Q (CHUL) Quebec University Hospital Research Centre &Laval University, 2705, Laurier Boulevard, Québec (QC) Canada G1V 4G2.
authors.forename: Rashmi G
authors.initials: RG
authors.lastname: Shah
authors.affiliation: New York University, Department of Chemistry, New York, NY, USA.
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Geacintov
authors.affiliation: Laboratory for Skin Cancer Research, CHU-Q (CHUL) Quebec University Hospital Research Centre &Laval University, 2705, Laurier Boulevard, Québec (QC) Canada G1V 4G2.
authors.forename: Girish M
authors.initials: GM
authors.lastname: Shah
mesh_terms: D055162:Biocatalysis / Q000528:radiation effects; D011232:Chemical Precipitation; D004247:DNA / Q000378:metabolism; D004249:DNA Damage; D018983:DNA Footprinting; D004260:DNA Repair / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D011065:Poly(ADP-ribose) Polymerases / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000528:radiation effects; D000072417:Protein Domains; D011740:Pyrimidine Dimers / Q000378:metabolism; D015203:Reproducibility of Results; D014466:Ultraviolet Rays
chemical_list: C117669:DDB2 protein, human; D004268:DNA-Binding Proteins; D011740:Pyrimidine Dimers; C058406:pyrimidine-pyrimidone dimer; D049452:Green Fluorescent Proteins; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases; D004255:Deoxyribodipyrimidine Photo-Lyase