Expression, Purification, and Characterization of Ultraviolet DNA Endonuclease from Schizosaccharomyces pombe

Abstract

Ultraviolet damage endonuclease (UVDE) is a 68.7 kDa DNA repair enzyme of Schizosaccharomyces pombe that recognizes cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4 PPs). UVDE is thought to initiate the first step in an alternative excision repair pathway for removal of UV light-induced DNA damage. We have overexpressed Delta228-UVDE, an active truncated form of UVDE, and have purified this protein to apparent homogeneity. We have characterized purified Delta228-UVDE with respect to its physical properties, divalent cation requirements, and kinetic parameters on oligodeoxynucleotide substrates containing a single CPD. DNA strand cleavage analysis indicates that both full-length UVDE and Delta228-UVDE incise the CPD-containing strand immediately 5' to the lesion. These results provide further insight into the UVDE-mediated alternative excision repair pathway.