Activity assays for poly-ADP ribose polymerase.
Poly(ADP-ribose) polymerase (PARP-1) is a nuclear enzyme that has traditionally been thought to require discontinuous or "damaged" DNA (dcDNA) as a coenzyme, a preconception that has limited research mainly to its role in cell pathology, i.e., DNA repair and apoptosis. Recent evidence has shown that this enzyme is broadly involved in normal cell physiological functions including chromatin modeling and gene regulation when DNA strand breaks are absent. We have recently shown that double-stranded DNA (dsDNA) serves as a more efficient coenzyme for PARP-1 than dcDNA, providing a mechanistic basis for PARP-1 function in normal cell physiology. Here we provide a detailed outline of methods for analyzing PARP-1 enzymatic activity using dsDNA as a coenzyme compared with broken or damaged DNA. Two procedures are described, one for analysis of auto-, and the other for trans-ADP-ribosylation. These assays provide a means of investigating the physiological role(s) of PARP-1 in normal cells.
Department of Anatomy, Cardiovascular Research Institute, School of Medicine, University of California, San Francisco, USA.
Eva
E
Kirsten
Ernest
E
Kun
Jerome
J
Mendeleyev
Charles P
CP
Ordahl
D002412:Cations; D042002:Chromatin Assembly and Disassembly; D004247:DNA / Q000378:metabolism; D004261:DNA Replication; D008967:Molecular Biology / Q000379:methods*; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis* / Q000378:metabolism* / Q000502:physiology; D011499:Protein Processing, Post-Translational; D012995:Solubility; D014158:Transcription, Genetic
D002412:Cations; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases