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 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.
Kaur
B
B
Department of Biochemistry, Division of Cancer Biology, Radiation and Oncology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Avery
A M
AM
Doetsch
P W
PW
D004260:DNA Repair; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D014466:Ultraviolet Rays
D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; D012965:Sodium Chloride; D004706:Endodeoxyribonucleases; C105073:uve1 protein, S pombe