CHARACTERIZATION OF A GAIN-OF-FUNCTION MUTANT OF POLY(ADP-RIBOSE) POLYMERASE

In order to examine the stucture-function relationship of the poly (AD P-ribose) polymerase (PARP) catalytic domain, potential active-site re sidues in the catalytic domain have previously been described. Here, w e have used mutagenesis with hydroxylamine to generate a random librar y of PARP mutants. The identification, overproduction in insect cells, purification and characterization of a gain-of-function mutant (L713F ) is described. We show that the k(cat) of this mutant is increased ov er nine times compared to the wild-type enzyme; the K-m for NAD(+) is unchanged. The size and the branching structure of the ADP-ribose poly mers are similar in both the wild-type and the mutant enzyme. This mut ation may have an allosteric effect on the catalytic site and could be useful in analyzing the consequences of poly ADP-ribose overproductio n in vivo on cell survival following DNA damage. (C) 1995 Academic Pre ss, Inc.