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 (K(D) = 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 (K(D) = ~2 x 10-8). Majority of the T[6-4]T-(6-4) photolyase complex dissociates very slowly (k(off) = 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 (φ) 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.