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

Biochem Biophys Res Commun. 1995 Jul 17;212(2):317-25. doi: 10.1006/bbrc.1995.1972.


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.

MeSH terms

  • 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


  • Hydroxylamines
  • NAD
  • Hydroxylamine
  • DNA
  • Poly(ADP-ribose) Polymerases