Nonisotopic methods for determination of poly(ADP-ribose) levels and detection of poly(ADP-ribose) polymerase.
Poly(ADP-ribosyl)ation is a post-translational modification catalyzed mostly by the 116-kDa enzyme poly(ADP-ribose) polymerase-1 (PARP-1), a nuclear enzyme that transfers an ADP-ribose moiety onto a limited number of nuclear proteins, including itself. When cells are exposed to environmental stresses such as alkylating agents or free radicals, there is up to a 500-fold increase in net poly(ADP-ribose) synthesis in response to DNA strand breaks. The enzyme responsible for 80% to 90% of this stimulated poly(ADP-ribose) synthesis is PARP-1, while other PARPs are responsible for the remaining 10% to 20%. The physiological meaning of these phenomena is not clear; however, it can be interpreted as a way of translating an event occurring on DNA to the nucleus by protein modification and finally to the cytoplasm via NAD(+) depletion. It has also been proposed that the presence of negatively charged poly(ADP-ribose) at the site of DNA damage may play several roles in regulation of base excision repair, p53 functions, and apoptosis. This unit describes protocols for measuring the levels of poly(ADP-ribose) in cells using nonisotopic reagents and for identifying the poly(ADP-ribose) polymerase enzymes present in cells.
Haince
Jean François
JF
Laval University Medical Research Center, Ste.-Foy, Quebec, Canada.
Poirier
Guy G
GG
Kirkland
Jim B
JB
D000818:Animals; D016923:Cell Death; D002478:Cells, Cultured; D019411:Clinical Laboratory Techniques; D004586:Electrophoresis; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D007150:Immunohistochemistry; D051379:Mice; D011064:Poly Adenosine Diphosphate Ribose / Q000032:analysis*; D011065:Poly(ADP-ribose) Polymerases / Q000032:analysis* / Q000737:chemistry
D011064:Poly Adenosine Diphosphate Ribose; D011065:Poly(ADP-ribose) Polymerases