Photoreactivating enzyme for (6-4) photoproducts in cultured goldfish cells.

We previously reported that when cultured goldfish cells are illuminated with fluorescent light, photorepair ability for both cyclobutane pyrimidine dimers and (6-4) photoproducts increased. In the present study, it was found that the duration of the induced photorepair ability for cyclobutane pyrimidine dimers was longer than that for (6-4) photoproducts, suggesting the presence of different photolyases for repair of these two major forms of DNA damage. A gel shift assay was then performed to show the presence of protein(s) binding to (6-4) photoproducts and its dissociation from (6-4) photoproducts under fluorescent light illumination. In addition, at 8 h after fluorescent light illumination of the cell, the binding of protein(s) to (6-4) photoproducts increased. The restriction enzymes that have recognition sites containing TT or TC sequences failed to digest the UV-irradiated DNA photoreactivated by using Escherichia coli photolyase for cyclobutane pyrimidine dimers, indicating that restriction enzymes could not function because (6-4) photoproducts remained in recognition sites. But, when UV-irradiated DNA depleted of cyclobutane pyrimidine dimers was incubated with extract of cultured goldfish cells under fluorescent light illumination, it was digested with those restriction enzymes. These results suggested the presence of (6-4) photolyase in cultured goldfish cells as in Drosophila, Xenopus and Crotalus.