Purification and characterization of the herpes simplex virus type 2-encoded uracil-DNA glycosylase.
The herpes simplex virus type 2 (HSV-2) uracil-DNA glycosylase (UNG) was from the nuclei of infected KB cells using ion exchange, affinity, and chromatofocusing chromatography techniques. Chromatography on DNA cellulose revealed two distinct HSV-2-encoded UNGs. One species, designated A, was purified approximately 324-fold, while the second species, designated B, was purified approximately 130-fold. The HSV UNG species B was observed to unidirectionally convert to the A species, suggesting that the B species binding to DNA cellulose may be the result of an association with other DNA binding proteins. SDS-PAGE demonstrated that both species A and B had molecular weights of 32,000. The HSV-2-encoded UNGs could be distinguished from the cellular nuclear UNG based upon differences in their behavior on the chromatography matrixes and by their molecular weights. There were no significant differences in the biochemical properties of the HSV-2-encoded or nuclear UNGs. Furthermore, all of these UNGs reacted with a monoclonal antibody produced against the human placental UNG. These data support recent studies, at both the DNA and the amino acid levels, which have demonstrated that this enzyme is highly conserved between species.
Department of Medical Microbiology and Immunology, Ohio State University, Columbus 43210.
T A
TA
Winters
M V
MV
Williams
D015153:Blotting, Western; D002460:Cell Line; D002845:Chromatography; D045647:DNA Glycosylases; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D009699:N-Glycosyl Hydrolases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018139:Simplexvirus / Q000201:enzymology*; D013379:Substrate Specificity; D051981:Uracil-DNA Glycosidase
D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase