Affinity Labeling of Flap-endonuclease FEN-1 by Photoreactive DNAs

D. Yu. Khlimankov¹, N. I. Rechkunova¹, D. M. Kolpashchikov¹, S. N. Khodyreva^1,2, and O. I. Lavrik^1,2*

¹Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, pr. Akademika Lavrent'eva 8, Novosibirsk, 630090 Russia; fax: (3832) 333-677; E-mail: lavrik@niboch.nsc.ru

²Novosibirsk State University, ul. Pirogova 2, Novosibirsk, 630090 Russia; fax: (3832) 397-101; E-mail: grant@fen.nsu.ru

^* To whom correspondence should be addressed.

Received November 16, 2000; Revision received March 1, 2001

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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.

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KEY WORDS: photoaffinity labeling, protein-nucleic acid interaction, flap-endonuclease, DNA repair

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