Further characterization of HeLa DNA polymerase ∈

Abstract

DNA polymerase ∈ (pol ∈) from HeLa cells was purified to near homogeneity, utilizing Mono S fast protein liquid chromatography for complete separation from pol α. The purified pol ∈ preparation showed two polypeptides of >200 and 55 kDa and a small amount of active 122-kDa proteolysis product on denaturing polyacrylamide gels. Pol ∈ (as well as pols α and δ) 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 ∈ activity, processivity, or substrate specificity. The size of the pol ∈ 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 ∈ transcript in HeLa cells or fibroblasts was seen after ultraviolet irradiation. Mouse polyclonal antiserum was produced to a 144-amino acid fragment of pol ∈ fused to staphylococcal protein A. This non-neutralizing polyclonal antiserum specifically recognized the catalytic subunit of pol ∈ by immunoblotting, but not that of pol α, β, or δ. In addition, mouse polyclonal antiserum raised against column-purified pol ∈ was able to recognize and to neutralize pol ∈, and a mouse monoclonal antibody was raised which was able to recognize specifically the catalytic subunit of pol ∈.