UNIT 18.7 Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of Poly(ADP-Ribose) Polymerase
Published Online: 1 FEB 2004
Copyright © 2003 by John Wiley and Sons, Inc.
Lab Protocol Title
Current Protocols in Cell Biology
How to Cite
Haince, J. F., Poirier, G. G. and Kirkland, J. B. 2004. Nonisotopic Methods for Determination of Poly(ADP-Ribose) Levels and Detection of Poly(ADP-Ribose) Polymerase. Current Protocols in Cell Biology. 21:18.7:18.7.1–18.7.26.
- Published Online: 1 FEB 2004
- Published Print: DEC 2003
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.
- DNA damage