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

Nupur K. Purohit, Mihaela Robu, Rashmi G. Shah, Nicholas Geacintov, Girish M. Shah

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Article number19020
JournalScientific Reports
Volume6
DOIs
StatePublished - Jan 12 2016

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DNA
In Vitro Techniques
Poly (ADP-Ribose) Polymerase-1
Housekeeping
Oligonucleotides
DNA Damage
Nucleotides
Enzymes

ASJC Scopus subject areas

  • General

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Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro. / Purohit, Nupur K.; Robu, Mihaela; Shah, Rashmi G.; Geacintov, Nicholas; Shah, Girish M.

In: Scientific Reports, Vol. 6, 19020, 12.01.2016.

Research output: Contribution to journalArticle

Purohit, Nupur K. ; Robu, Mihaela ; Shah, Rashmi G. ; Geacintov, Nicholas ; Shah, Girish M. / Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro. In: Scientific Reports. 2016 ; Vol. 6.
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