EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF ULTRAVIOLET DNA ENDONUCLEASE FROM SCHIZOSACCHAROMYCES-POMBE

Ultraviolet damage endonuclease (UVDE) is a 68.7 kDa DNA repair enzyme of Schizosaccharomyces pombe that recognizes cyclobutane pyrimidine d imers (CPDs) and 6-4 photoproducts (6-4 PPs), UVDE is thought to initi ate the first step in an alternative excision repair pathway for remov al of UV light-induced DNA damage. We have overexpressed Delta 228-UVD E, an active truncated form of UVDE, and have purified this protein to apparent homogeneity. We have characterized purified Delta 228-UVDE w ith respect to its physical properties, divalent cation requirements, and kinetic parameters on oligodeoxynucleotide substrates containing a single CFD. DNA strand cleavage analysis indicates that both full-len gth UVDE and Delta 228-UVDE incise the CPD-containing strand immediate ly 5' to the lesion. These results provide further insight into the UV DE-mediated alternative excision repair pathway.