Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro

• Published in: Scientific reports 2016-01, Vol.6 (1), p.19020-19020, Article 19020
• Main Authors: Purohit, Nupur K., Robu, Mihaela, Shah, Rashmi G., Geacintov, Nicholas E., Shah, Girish M.
• Format: Article
• Language: English
• Subjects: 14/1; 14/35; 14/63; 38/22; 38/71; 42/35; 45/22; 45/29; 631/337/1427/1430; 631/45/607/1159; 631/80/458/2389; 82/1; 82/51; Adenosine diphosphate; Biocatalysis - radiation effects; Chemical Precipitation; Deoxyribodipyrimidine Photo-Lyase - metabolism; Deoxyribonucleic acid; DNA; DNA - metabolism; DNA Damage; DNA Footprinting; DNA repair; DNA Repair - radiation effects; DNA-Binding Proteins - metabolism; Fractionation; Green Fluorescent Proteins - metabolism; Humanities and Social Sciences; Humans; Irradiation; Lesions; Models, Biological; multidisciplinary; Nucleotides; Oligonucleotides; Poly(ADP-ribose) polymerase; Poly(ADP-ribose) Polymerases - chemistry; Poly(ADP-ribose) Polymerases - metabolism; Protein Binding - radiation effects; Protein Domains; Pyrimidine Dimers - metabolism; Reproducibility of Results; Ribose; Science; Ultraviolet Rays
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/srep19020

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.