Role of the tryptophan residue in the vicinity of the catalytic center of exonuclease III family AP endonucleases: AP site recognition mechanism.
The mechanisms by which AP endonucleases recognize AP sites have not yet been determined. Based on our previous study with Escherichia coli exonuclease III (ExoIII), the ExoIII family AP endonucleases probably recognize the DNA-pocket formed at an AP site. The indole ring of a conserved tryptophan residue in the vicinity of the catalytic site presumably intercalates into this pocket. To test this hypothesis, we constructed a series of mutants of ExoIII and human APE1. Trp-212 of ExoIII and Trp-280 of APE1 were critical to the AP endonuclease activity and binding to DNA containing an AP site. To confirm the ability of the tryptophan residue to intercalate with the AP site, we examined the interaction between an oligopeptide containing a tryptophan residue and an oligonucleotide containing AP sites, using spectrofluorimetry and surface plasmon resonance (SPR) technology. The tryptophan residue of the oligopeptide specifically intercalated into an AP site of DNA. The tryptophan residue in the vicinity of the catalytic site of the ExoIII family AP endonucleases plays a key role in the recognition of AP sites.
Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan.
Kohichi
K
Kaneda
Junichi
J
Sekiguchi
Toshio
T
Shida
D000595:Amino Acid Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D005090:Exodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D016415:Sequence Alignment; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry*
D010455:Peptides; D014364:Tryptophan; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase