Further characterization of HeLa DNA polymerase epsilon.

The Journal of Biological Chemistry
G S Chui, S Linn

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-neutralizin...Continue Reading

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Citations

Sep 13, 2000·Nucleic Acids Research·N VlatkovicM T Boyd
Nov 3, 2010·Nucleic Acids Research·Dagmara A KoronaZachary F Pursell
Mar 16, 2007·Journal of Virology·Kevin NashNicholas Muzyczka
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Oct 17, 2012·PLoS Genetics·Scott A LujanThomas A Kunkel
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May 28, 2014·IUBMB Life·Erin E Henninger, Zachary F Pursell
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