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

• Published in: Biochemistry (Moscow) Vol. 66; no. 7; pp. 733 - 739
• Main Authors: Khlimankov, D Y, Rechkunova, N I, Kolpashchikov, D M, Khodyreva, S N, Lavrik, O I
• Format: Journal Article
• Language: English
• Published: United States Springer Nature B.V 01.07.2001
• Subjects: Animals; Deoxyribonucleic acid; DNA; DNA Polymerase beta - metabolism; DNA Primers - genetics; DNA Primers - metabolism; DNA Repair; DNA Replication; Endodeoxyribonucleases - chemistry; Endodeoxyribonucleases - metabolism; Fibroblasts - metabolism; Flap Endonucleases; Labeling; Mice; Oligonucleotides - chemistry; Oligonucleotides - metabolism; Photoaffinity Labels - chemistry; Photoaffinity Labels - metabolism; Substrate Specificity
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1023/A:1010204527130

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.