Processing of UV damage in vitro by FEN-1 proteins as part of an alternative DNA excision repair pathway

Ultraviolet (UV) irradiation induces predominantly cyclobutane and (6-4) py rimidine dimer photoproducts in DNA. Several mechanisms for repairing these mutagenic UV-induced DNA lesions have been identified. Nucleotide excision repair is a major pathway, but mechanisms involving photolyases and DNA gl ycosylases have also been characterized. Recently, a novel UV damage endonu clease (UVDE) was identified that initiates an excision repair pathway diff erent from previously established repair mechanisms. Homologues of UVDE hav e been found in eukaryotes as well as in bacteria. In this report, we have used oligonucleotide substrates containing site-specific cyclobutane pyrimi dine dimers and (6-4) photoproducts for the characterization of this UV dam age repair pathway. After introduction of single strand breaks at the 5' si des of the photolesions by UVDE, these intermediates became substrates for cleavage by flap endonucleases (FEN-1 proteins). FEN-1 homologues from huma ns, Saccharomyces cerevisiae, and Schizosaccharomyces pombe all cleaved the UVDE-nicked substrates at similar positions 3' to the photolesions. T4 end onuclease V-incised DNA was processed in the same way. Both nicked and flap ped DNA substrates with photolesions (the latter may be intermediates in DN A polymerase-catalyzed strand displacement synthesis) were cleaved by FEN-1 . The data suggest that the two enzymatic activities, UVDE and FEN-1, are p art of an alternative excision repair pathway for repair of UV photoproduct s.