Affinity labeling of flap-endonuclease FEN-1 by photoreactive DNAs

Biochemistry (Mosc). 2001 Jul;66(7):733-9. doi: 10.1023/a:1010204527130.

Abstract

Eukaryotic flap-endonuclease (FEN-1) is 42-kD single-subunit structure-specific nuclease that cleaves 5'-flap strands of the branched DNA structure and possesses 5'-exonuclease activity. FEN-1 participates in DNA replication, repair, and recombination. The interaction of FEN-1 with DNA structures generated during replication and repair was studied using two types of photoreactive oligonucleotides. Oligonucleotides bearing a photoreactive arylazido group at the 3'-end of the primer were synthesized in situ by the action of DNA polymerase beta using base-substituted photoreactive dUTP analogs as the substrates. The photoreactive group was also bound to the 5'-end phosphate group of the oligonucleotide by chemical synthesis. Interaction of FEN-1 with both 5'- and 3'-ends of the nick or with primer-template systems containing 5'- or 3'-protruding DNA strands was shown. Formation of a structure with the 5'-flap containing the photoreactive group results in decrease of the level of protein labeling caused by cleavage of the photoreactive group due to FEN-1 endonuclease activity. Photoaffinity labeling of proteins of mouse fibroblast cell extract was performed using the radioactively labeled DNA duplex with the photoreactive group at the 3'-end and the apurine/apyrimidine site at the 5'-end of the nick. This structure is a photoreactive analog of an intermediate formed during DNA repair and was generated by the action of cell enzymes from the initial DNA duplex containing the 3-hydroxy-2-hydroxymethyltetrahydrofurane residue. FEN-1 is shown to be one of the photolabeled proteins; this indicates possible participation of this enzyme in base excision repair.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • DNA Polymerase beta / metabolism*
  • DNA Primers / genetics
  • DNA Primers / metabolism*
  • DNA Repair
  • DNA Replication
  • Endodeoxyribonucleases / chemistry
  • Endodeoxyribonucleases / metabolism*
  • Fibroblasts / metabolism
  • Flap Endonucleases
  • Mice
  • Oligonucleotides / chemistry
  • Oligonucleotides / metabolism*
  • Photoaffinity Labels / chemistry
  • Photoaffinity Labels / metabolism*
  • Substrate Specificity

Substances

  • DNA Primers
  • Oligonucleotides
  • Photoaffinity Labels
  • DNA Polymerase beta
  • Endodeoxyribonucleases
  • Flap Endonucleases