Characterization of a Gain-of-Function Mutant of Poly(ADP-Ribose) Polymerase
| Autor: | G. de-Murcia, J.M. Demurcia, F. Dantzer, M. Ofarrell, Everson Alves Miranda |
|---|---|
| Rok vydání: | 1995 |
| Předmět: |
DNA damage
Poly ADP ribose polymerase Mutant Biophysics Hydroxylamine Spodoptera Hydroxylamines Biochemistry Structure-Activity Relationship Allosteric Regulation Escherichia coli Animals Enzyme kinetics Overproduction Molecular Biology Polymerase chemistry.chemical_classification Binding Sites biology Gene Transfer Techniques DNA Cell Biology NAD Molecular biology Kinetics Enzyme chemistry Mutagenesis biology.protein NAD+ kinase Poly(ADP-ribose) Polymerases Baculoviridae DNA Damage |
| Zdroj: | Biochemical and Biophysical Research Communications. 212:317-325 |
| ISSN: | 0006-291X |
| DOI: | 10.1006/bbrc.1995.1972 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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