Characterization of a Gain-of-Function Mutant of Poly(ADP-Ribose) Polymerase

In order to examine the stucture-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. T...

Full description

Saved in:
Bibliographic Details
Published in:Biochemical and biophysical research communications 1995-07, Vol.212 (2), p.317-325
Main Authors: Miranda, E.A., Dantzer, F., Ofarrell, M., Demurcia, G., Demurcia, J.M.
Format: Article
Language:English
Subjects:
Allosteric Regulation
Animals
Baculoviridae - genetics
Binding Sites
DNA - chemistry
DNA - drug effects
DNA Damage
Escherichia coli - genetics
Gene Transfer Techniques
Hydroxylamine
Hydroxylamines - pharmacology
Kinetics
Mutagenesis
NAD - metabolism
Poly(ADP-ribose) Polymerases - chemistry
Poly(ADP-ribose) Polymerases - genetics
Poly(ADP-ribose) Polymerases - metabolism
Spodoptera - metabolism
Structure-Activity Relationship
Citations: Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:In order to examine the stucture-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.
ISSN:0006-291X
1090-2104
DOI:10.1006/bbrc.1995.1972