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.
Highlights
Appears to be required for long patch DNA repair in permeabilized cells (3)
Unlike pol 8, polymerase E (pol E) is highly processive in the absence of proliferating cell nuclear antigen (PCNA)
It has been suggested that inhibition of HeLa pol E by NaCl can be overcome by the presence of HSSB, AI, and PCNA (8)
Summary
Nucleotides and Nucleic Acids-Unlabeled deoxy- and ribonucleotides were purchased from Pharmacia Biotech Inc.; [a· 32PJdTTP and [a-32PJdATP were from Amersham Corp. The reaction mixture was extracted with phenol and chromatographed on Sephadex G-150 to remove residual NTPs. Enzymes, Proteins, and Plasmids-DNA pols ex, S, and e and PCNA were purified from HeLa cells as described (9). The glutathione S-transferase (GST) fusion vector, pGEX-lAT, was purchased from Pharmacia with the correct reading frame for the pol E fragment and maintained in E. coli DH5a. The pol E fragment used for constructing the Staphylococcus aureus protein A (SpA-) fusion protein was a 435-bp BamHIIEcoRI fragment of a 1.2-kb clone obtained in this laboratory. This pol E fragment (nucleotides 1611-2045) (14) contains the conserved region II of the a-class DNA polymerases (15, 16).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
