FURTHER CHARACTERIZATION OF HELA DNA-POLYMERASE-EPSILON

DNA polymerase epsilon (pol epsilon) from HeLa cells was purified to n ear homogeneity, utilizing Mono S fast protein liquid chromatography f or complete separation from pol alpha. The purified pol epsilon prepar ation 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 th is preparation's pol epsilon activity, processivity, or substrate spec ificity. The size of the pol epsilon transcript for the catalytic subu nit (>200 kDa) was investigated in both normal human fibroblasts and H eLa cells. A 7.7-kilobase transcript was detected which was 5-16-fold more prevalent in proliferating than in quiescent HeLa cells. No signi ficant difference in the level of pol epsilon transcript in HeLa cells or fibroblasts was seen after ultraviolet irradiation. Mouse polyclon al antiserum was produced to a 144-amino acid fragment of pol epsilon fused to staphylococcal protein A. This non-neutralizing polyclonal an tiserum specifically recognized the catalytic subunit of pol epsilon b y immunoblotting, but not that of pol alpha, beta, or delta. In additi on, mouse polyclonal antiserum raised against column-purified pol epsi lon was able to recognize and to neutralize pol epsilon, and a mouse m onoclonal antibody was raised which was able to recognize specifically the catalytic subunit of pol epsilon.