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.
Highlights
The abundance of poly (ADP-ribose) polymerase-1 (PARP-1) in mammalian cells and its rapid catalytic activation to form polymers of ADP-ribose (PAR) in the presence of various types of DNA damages with or without strand breaks has made it an ideal first responder at the lesion site to influence downstream events[1,2]
The second major challenge is that we do not know the exact footprint of PARP-1 at the UV-lesion site that could explain its interaction with different nucleotide excision repair (NER) proteins
We first determined whether different permeabilization-fixation protocols conventionally used for PARP-1 could reveal a direct recruitment of PARP-1 to UVC-induced DNA photolesions in situ
Summary
Novel in situ fractionation protocol to reveal recruitment of endogenous PARP-1 to UV-induced DNA lesion. Our results show that PARP-1 and DDB2 do not prevent access to the lesion site by other repair proteins such as CPD photolyase, and may even be more flexible and accommodating in the in vivo conditions as compared to our in vitro condition in which damaged DNA is anchored to beads and interacting proteins are cross-linked to the DNA. Together, these two novel assays will open new avenues to study the ever-expanding roles of PARP-1 in NER and housekeeping functions in which PARP-1 shows subtle interactions with DNA without strand breaks
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