The (6-4) photolyase catalyzes the photoreversal of the (6-4) dipyrimidine photoproducts induced in DNA by ultraviolet light. Using the cloned Drosophila melanogaster (6-4) photolyase gene, we overproduced and purified the recombinant enzyme. The binding and catalytic properties of the enzyme were investigated using natural substrates, T[6-4]T and T[6-4]C, and the Dewar isomer of (6-4) photoproduct and substrate analogs s5T[6-4]T/thietane, mes5T[6-4]T, and the N-methyl-3'T thietane analog of the oxetane intermediate. The enzyme binds to the natural substrates and to mes5T[6-4]T with high affinity (KD approximately 10(-9)-10(-10) M) and produces a DNase I footprint of about 20 base pairs around the photolesion. Several lines of evidence suggest that upon binding by the enzyme, the photoproduct flips out of the duplex. Of the four substrates that bind with high affinity to the enzyme, T[6-4]T and T[6-4]C are repaired with relatively high quantum yields compared with the Dewar isomer and the mes5T[6-4]T which are repaired with 300-400-fold lower quantum yield than the former two photoproducts. Reduction of the FAD cofactor with dithionite increases the quantum yield of repair. Taken together, the data are consistent with photoinduced electron transfer from reduced FAD to substrate, in a manner analogous to the cyclobutane pyrimidine dimer photolyase.