Photoaffinity labeling of T4 endonuclease V with a substrate containing a phenyldiazirine derivative.
T4 endonuclease V recognizes thymine photodimers in DNA duplexes and, in a two-step reaction, cleaves the glycosyl linkage of the 5'-side thymidine and the phosphodiester linkage. To determine the amino acid residues responsible for binding thymine photodimers, a photoaffinity reagent, 4-(1-azi-2,2,2-trifluoroethyl)-benzoate, was linked to the aminoalkylphosphonate of a thymine photodimer in a 14-mer duplex. The reactive substrate was treated with the enzyme under UV light (365 nm). The nascent enzyme and the modified enzyme were treated with lysyl endopeptidase, and the peptide maps were compared. Three peptides from the C terminus were found to interact with the reactive oligonucleotide to various extents. The three modified peptides were isolated and analyzed by Edman degradation. The amino acid residues Gly-133, Tyr-129, and Thr-89 were partially linked with the reactive substrate and may be involved in the binding of thymine photodimers.
Hori
N
N
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Iwai
S
S
Inoue
H
H
Ohtsuka
E
E
D000345:Affinity Labels; D000595:Amino Acid Sequence; D001389:Azirines; D001483:Base Sequence; D001565:Benzoates; D001665:Binding Sites; D003432:Cross-Linking Reagents; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010446:Peptide Fragments / Q000302:isolation & purification; D011740:Pyrimidine Dimers; D013604:T-Phages / Q000201:enzymology*; D014764:Viral Proteins
D000345:Affinity Labels; D001389:Azirines; D001565:Benzoates; D003432:Cross-Linking Reagents; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011740:Pyrimidine Dimers; D014764:Viral Proteins; C051320:4-(3-trifluoromethyldiazirino)benzoic acid; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer)