Characterization of a large form of DNA polymerase delta from HeLa cells that is insensitive to proliferating cell nuclear antigen.

Abstract:

A large form of DNA polymerase delta from HeLa cells was recently purified in this laboratory as a factor required for conservative DNA synthesis in a reconstituted system utilizing UV-irradiated permeabilized human diploid fibroblasts (Nishida, C., Reinhard, P., and Linn, S. (1988) J. Biol. Chem. 263, 501-510). We have now purified this form of the enzyme utilizing its polymerase activity and further characterized it. The enzyme activity sediments at 11.1 S in low salt and 6.8 S in high salt. In both cases, activity cosediments with the major visible peptide displayed by sodium dodecyl sulfate-polyacrylamide gels which has an Mr of 215,000. This value is consistent with the molecular mass calculated from the sedimentation coefficient and gel filtration behavior in high salt. In low salt the apparent molecular mass was approximately double. The enzyme prefers poly(dA).oligo(dT) as template/primer in low salt, with which it has a processivity of several thousand nucleotides in 1 mM MgCl2. At isotonic KCl or potassium phosphate concentrations, the preferred template/primer is activated DNA. Proliferating cell nuclear antigen, also characterized as a DNA polymerase delta auxiliary protein, does not increase the activity of this preparation of the enzyme. An antibody to the proliferating cell nuclear antigen has no inhibitory effect, nor is it able to recognize any peptides in immunoblots of purified enzyme fractions. Under polymerizing conditions, the enzyme removes mismatched, but not matched nucleotides from the 3' terminus of oligo(dT) annealed to poly(dA) suggesting a proofreading function. The properties of this form of DNA polymerase delta distinguish it from other preparations reported in the literature.

Polymerases:

Topics:

Historical Protein Properties (MW, pI, ...), Kinetic Parameters, Nucleotide Incorporation, Exonuclease Activity

Note:

switched from pol delta to pol epsilon due to change in nomenclature

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
Human Pol epsilon Syvaoja J1989 Molecular Weight 2.15E+05 Dalton Technique: SDS-PAGE
Human Pol epsilon Syvaoja J1989 3-5' Exonuclease (proofreading) Yes
Human Pol epsilon Syvaoja J1989 Cloned or native Native organism
Human Pol epsilon Syvaoja J1989 Tagged No
Human Pol epsilon Syvaoja J1989 Full length or truncated Full length
Human Pol epsilon Syvaoja J1989 Processivity 1kb
Human Pol epsilon Syvaoja J1989 Specific Activity 2.4E+04 units/mg
Human Pol epsilon Syvaoja J1989 KM 2.5uM Reaction: Nucleotide incorporation; Substrate: dTTP
Human Pol epsilon Syvaoja J1989 KM 3uM Reaction: Polymerase-DNA interaction; Substrate: DNA template
Human Pol epsilon Syvaoja J1989 KM 4.7uM Reaction: Nucleotide incorporation; Substrate: dNTPs

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