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

Eukaryotic flap-endonuclease (FEN-1) is 42-kD single-subunit structure-spec ific nuclease that cleaves 5 ' -flap strands of the branched DNA structure and possesses 5 ' -exonuclease activity. FEN-1 participates in DNA replicat ion, repair, and recombination. The interaction of FEN-1 with DNA structure s generated during replication and repair was studied using two types of ph otoreactive oligonucleotides, Oligonucleotides bearing a photoreactive aryl azido group at the 3 ' -end of the primer were synthesized in situ by the a ction of DNA polymerase beta using base -substituted photoreactive dUTP ana logs as the substrates. The photoreactive group was also bound to the 5 ' - end phosphate group of the oligonucleotide by chemical synthesis. Interacti on of FEN-1 with both 5 '- and 3 ' -ends of the nick or with primer-templat e systems containing 5 '- or 3 ' -protruding DNA strands was shown. Formati on of a structure with the 5'-flap containing the photoreactive group resul ts in decrease of the level of protein labeling caused by cleavage of the p hotoreactive group due to FEN-1 endonuclease activity. Photoaffinity labeli ng of proteins of mouse fibroblast cell extract was performed using the rad ioactively labeled DNA duplex with the photoreactive group at the 3 ' -end and the apurine/apyrimidine site at the 5 ' -end of the nick. This structur e 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 co ntaining the 3-hydroxy-2-hydroxymethyltetrahydrofurane residue. FEN-1 is sh own to be one of the photolabeled proteins; this indicates possible partici pation of this enzyme in base excision repair.