Further characterization of HeLa DNA polymerase epsilon.
DNA polymerase epsilon (pol epsilon) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol alpha. The purified pol epsilon preparation showed two polypeptides of > 200 and 55 kDa and a small amount of active 122-kDa proteolysis product on denaturing polyacrylamide gels. Pol epsilon (as well as pols alpha and delta) is optimally active in 100-150 mM potassium glutamate and 15 mM MgCl2. Replication factors RF-A and RF-C, proliferating cell nuclear antigen, and Escherichia coli single-stranded DNA binding protein showed no significant effect on this preparation's pol epsilon activity, processivity, or substrate specificity. The size of the pol epsilon transcript for the catalytic subunit (> 200 kDa) was investigated in both normal human fibroblasts and HeLa cells. A 7.7-kilobase transcript was detected which was 5-16-fold more prevalent in proliferating than in quiescent HeLa cells. No significant difference in the level of pol epsilon transcript in HeLa cells or fibroblasts was seen after ultraviolet irradiation. Mouse polyclonal antiserum was produced to a 144-amino acid fragment of pol epsilon fused to staphylococcal protein A. This non-neutralizing polyclonal antiserum specifically recognized the catalytic subunit of pol epsilon by immunoblotting, but not that of pol alpha, beta, or delta. In addition, mouse polyclonal antiserum raised against column-purified pol epsilon was able to recognize and to neutralize pol epsilon, and a mouse monoclonal antibody was raised which was able to recognize specifically the catalytic subunit of pol epsilon.
Chui
G
G
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3202, USA.
Linn
S
S
D000818:Animals; D000911:Antibodies, Monoclonal; D002384:Catalysis; D004257:DNA Polymerase II; D004261:DNA Replication; D004259:DNA-Directed DNA Polymerase / Q000276:immunology / Q000378:metabolism*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D012313:RNA / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000378:metabolism; D013698:Templates, Genetic
D000911:Antibodies, Monoclonal; D011993:Recombinant Fusion Proteins; D013205:Staphylococcal Protein A; D012313:RNA; D004257:DNA Polymerase II; D004259:DNA-Directed DNA Polymerase