Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells

EMBO J. 1993 May;12(5):2109-17.

Abstract

The zinc-finger DNA-binding domain (DBD) of poly (ADP-ribose) polymerase (PARP, EC 2.4.2.30) specifically recognizes DNA strand breaks induced by various DNA-damaging agents in eukaryotes. This, in turn, triggers the synthesis of polymers of ADP-ribose linked to nuclear proteins during DNA repair. The 46 kDa DBD of human PARP, and several derivatives thereof mutated in its first or second zinc-finger, were overproduced in Escherichia coli, in CV-1 monkey cells or in human fibroblasts to study their DNA-binding properties, the trans-dominant inhibition of resident PARP activity, and the consequences on DNA repair, respectively. A positive correlation was found between the in vitro DNA-binding capacity of the recombinant DBD polypeptides and their inhibitory effect on PARP activity stimulated by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Furthermore, overproduced wild-type DBD blocked unscheduled DNA synthesis induced in living cells by MNNG treatment, but not that induced by UV irradiation. These results define a critical role for the second zinc-finger of PARP for DNA single-stranded break binding and furthermore underscore the importance for PARP to act as a critical regulatory component in the repair of DNA damage induced by alkylating agents.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkylation
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cell Line
  • Cells, Cultured
  • DNA / biosynthesis*
  • DNA / drug effects
  • DNA / metabolism
  • DNA Repair*
  • Escherichia coli
  • Haplorhini
  • Humans
  • Methylnitronitrosoguanidine / pharmacology
  • Molecular Sequence Data
  • Mutation
  • Poly(ADP-ribose) Polymerase Inhibitors
  • Poly(ADP-ribose) Polymerases / genetics
  • Poly(ADP-ribose) Polymerases / metabolism*
  • Recombinant Proteins / metabolism
  • Transfection
  • Zinc Fingers

Substances

  • Poly(ADP-ribose) Polymerase Inhibitors
  • Recombinant Proteins
  • Methylnitronitrosoguanidine
  • DNA
  • Poly(ADP-ribose) Polymerases