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Biochem Biophys Res Commun. 1995 Jul 17;212(2):317-25.

Characterisation of a gain-of-function mutant of poly(ADP-ribose) polymerase.

Author information

1
Ecole Supérieure de Biotechnologie de Strasbourg, UPR 9003 du Centre National de la Recherche Scientifique, Illkirch Graffenstaden, France.

Abstract

In order to examine the structure-function relationship of the poly (ADP-ribose) polymerase (PARP) catalytic domain, potential active-site residues in the catalytic domain have previously been described. Here, we have used mutagenesis with hydroxylamine to generate a random library 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 kcat of this mutant is increased over nine times compared to the wild-type enzyme; the Km for NAD+ is unchanged. The size and the branching structure of the ADP-ribose polymers are similar in both the wild-type and the mutant enzyme. This mutation may have an allosteric effect on the catalytic site and could be useful in analyzing the consequences of poly ADP-ribose overproduction in vivo on cell survival following DNA damage.

PMID:
7626044
DOI:
10.1006/bbrc.1995.1972
[Indexed for MEDLINE]

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