Immunocytochemical localization of the base excision repair enzyme uracil DNA glycosylase in quiescent and proliferating normal human cells.
The immunocytochemical localization of the base excision repair enzyme uracil DNA glycosylase was examined as a function of cell proliferation. Two nontransformed normal human fibroblast cell strains were analyzed using an anti-human uracil DNA glycosylase monoclonal antibody. In quiescent cells, basal levels of a nonnuclear immunocytochemically reactive glycosylase protein were detected. No nuclear immunofluorescence was observed. In contrast, in proliferating cells, intense immunofluorescence could be detected exclusively in the nuclear or perinuclear regions. As proliferation diminished, basal levels of the nonnuclear immunocytochemically reactive glycosylase were again observed. The subcellular distribution of the glycosylase was examined in parallel by in vitro biochemical assay. In quiescent cells, glycosylase activity was observed in both the nuclear and membrane fractions. A small amount of enzyme activity could be detected in the soluble cytoplasmic fraction. Immunoblot analysis demonstrated a Mr 37,000 glycosylase protein in each subcellular fraction. During cell proliferation, there was an increase in glycosylase activity in each of the subcellular fractions. These results suggest a correlation between the proliferative state of normal human cells and the preferential nuclear or perinuclear localization of an immunocytochemically reactive glycosylase protein. Further, immunofluorescence of the nuclear enzyme may be dependent on defined conformational states of that nuclear glycosylase in the cell cycle.
Cool
B L
BL
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
Sirover
M A
MA
D000974:Antibodies, Antinuclear; D002455:Cell Division; D002467:Cell Nucleus / Q000201:enzymology*; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis*; D045647:DNA Glycosylases; D004260:DNA Repair; D006801:Humans; D009699:N-Glycosyl Hydrolases / Q000032:analysis*; D051981:Uracil-DNA Glycosidase
D000974:Antibodies, Antinuclear; D004247:DNA; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase