Further characterization of HeLa DNA polymerase epsilon.

Abstract:

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.

Polymerases:

Topics:

Source / Purification, Historical Protein Properties (MW, pI, ...), Nucleotide Incorporation, Modulators/Inhibitors, Other Enzymatic Activities

One line summary:

Pol epsilon activity does not depend on PCNA. Pol epsilon does extend from RNA primers.

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Human Pol epsilon Chui G1995 Cloned or native Native organism
Human Pol epsilon Chui G1995 Tagged No
Human Pol epsilon Chui G1995 Full length or truncated Full length
Human Pol epsilon Chui G1995 Extension from RNA primer Yes
Human Pol epsilon Chui G1995 Percent/Fold Effect 0 % decrease Reaction: Nucleotide incorporation

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