Photoaffinity labeling of mouse fibroblast enzymes by a base excision repair intermediate. Evidence for the role of poly(ADP-ribose) polymerase-1 in DNA repair

J Biol Chem. 2001 Jul 6;276(27):25541-8. doi: 10.1074/jbc.M102125200. Epub 2001 May 4.


To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta, and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Binding, Competitive
  • Cell Line
  • DNA Repair*
  • Deoxycytosine Nucleotides / metabolism
  • Fibroblasts / enzymology*
  • Humans
  • In Situ Nick-End Labeling
  • Isoenzymes / metabolism*
  • Methyl Methanesulfonate / pharmacology
  • Mice
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Photoaffinity Labels / metabolism*
  • Poly(ADP-ribose) Polymerases / metabolism*
  • Spectrophotometry, Ultraviolet


  • Deoxycytosine Nucleotides
  • Isoenzymes
  • Photoaffinity Labels
  • 2'-deoxycytidine 5'-triphosphate
  • Methyl Methanesulfonate
  • Poly(ADP-ribose) Polymerases