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Anal Biochem. 1992 Jun;203(2):252-8.

Poly(ADP-ribose) synthesis and degradation in mammalian nuclei.

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Linus Pauling Institute of Science and Medicine, Palo Alto, California 94306.


Poly(ADP-ribose) built from NAD+ on histones and other nuclear proteins by poly(ADP-ribose) polymerase is involved in repair, replication, gene expression, recombination, and chromatin remodeling in embryogenesis. Such nuclear processes are believed to be facilitated by opening up of condensed chromatin structures and by removal of histones from DNA at damaged sites as well as at origins of replication and transcription initiation sites. In addition, poly(ADP-ribosyl)ation might be involved in the up or down regulation of the activity of key nuclear enzymes. Poly(ADP-ribose) is rapidly synthesized at sites containing DNA strand breaks and is then rapidly degraded (half-life 0.5-5 min) by poly(ADP-ribose)glycohydrolase. High-resolution polyacrylamide gel electrophoresis is used in this study to analyze the rate of consumption of [32P]NAD+, the rate of formation of poly(ADP-ribose) molecules, and the rate of appearance of ADP-ribose, AMP, and phosphoribosyl-AMP, the catabolites of poly(ADP-ribose) in isolated nuclei from mouse cells in culture. Our method permits direct loading of aliquots of nuclei at time intervals on the polyacrylamide gel. The action of poly(ADP-ribose) glycohydrolase that degrades the polymer starts at less than 2 min from polymer formation. A poly(ADP-ribose) phosphodiesterase present in mammalian cell nuclei begins degrading poly(ADP-ribose) or unincorporated NAD+ and free ADP-ribose at 10 min. Mammalian phosphodiesterase is identified as an enzyme more important than previously thought which might degrade poly(ADP-ribosyl)ated proteins but also recycle the ADP-ribose produced from di- to poly(ADP-ribosyl)ated proteins by glycohydrolase into utilizable AMP units.

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