title: Binding and catalytic properties of Xenopus (6-4) photolyase.
abstract: Xenopus (6-4) photolyase binds with high affinity to DNA bearing a (6-4) photoproduct and repairs it in a light-dependent reaction. To clarify its repair mechanism of (6-4) photolyase, we determined its binding and catalytic properties using synthetic DNA substrate which carries a photoproduct at a single location. The (6-4) photolyase binds to T[6-4]T in double-stranded DNA with high affinity (KD = 10(-9)) and to T[6-4]T in single-stranded DNA and T[Dewar]T in double- and single-stranded DNA although with slightly lower affinity (KD = approximately 2 x 10(-8)). Majority of the T[6-4]T-(6-4) photolyase complex dissociates very slowly (koff = 2.9 x 10(-5) s-1). Its absolute action spectrum without a second chromophore in the 350-600 nm region closely matches the absorption spectrum of the enzyme. The quantum yield (phi) of repair is approximately 0.11. The fully reduced form (E-FADH-) of (6-4) photolyase is catalytically active. Direct analysis of the photoreactivated product showed that (6-4) photolyase restores the original pyrimidines. These findings demonstrate that cis, syn-cyclobutane pyrimidine dimer photolyase and (6-4) photolyase are quite similar, but they are different with regard to the binding properties.
authors.affiliation: Radiation Biology Center, Kyoto University, Yoshida konoe-cho, Sakyo-ku, Kyoto 606-01, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Hitomi
authors.forename: S T
authors.initials: ST
authors.lastname: Kim
authors.forename: S
authors.initials: S
authors.lastname: Iwai
authors.forename: N
authors.initials: N
authors.lastname: Harima
authors.forename: E
authors.initials: E
authors.lastname: Otoshi
authors.forename: M
authors.initials: M
authors.lastname: Ikenaga
authors.forename: T
authors.initials: T
authors.lastname: Todo
mesh_terms: D000818:Animals; D002384:Catalysis; D018983:DNA Footprinting; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D004331:Drosophila melanogaster; D011485:Protein Binding; D013379:Substrate Specificity; D014981:Xenopus
chemical_list: D003850:Deoxyribonuclease I; C098727:pyrimidine(6-4)pyrimidone photolyase; D004255:Deoxyribodipyrimidine Photo-Lyase