Affinity labeling of flap-endonuclease FEN-1 by photoreactive DNAs.
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.
Khlimankov
D Y
DY
Novosibirsk Institute of Bioorganic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russia.
Rechkunova
N I
NI
Kolpashchikov
D M
DM
Khodyreva
S N
SN
Lavrik
O I
OI
D000818:Animals; D019951:DNA Polymerase beta / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism*; D004260:DNA Repair; D004261:DNA Replication; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D045585:Flap Endonucleases; D051379:Mice; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D019920:Photoaffinity Labels / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D017931:DNA Primers; D009841:Oligonucleotides; D019920:Photoaffinity Labels; D019951:DNA Polymerase beta; D004706:Endodeoxyribonucleases; D045585:Flap Endonucleases