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PMID:12571237

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Citation

Chilkova, O, Jonsson, BH and Johansson, E (2003) The quaternary structure of DNA polymerase epsilon from Saccharomyces cerevisiae. J. Biol. Chem. 278:14082-6

Abstract

DNA polymerase epsilon (Pol epsilon) from Saccharomyces cerevisiae consists of four subunits (Pol2, Dpb2, Dpb3, and Dpb4) and is essential for chromosomal DNA replication. Biochemical characterizations of Pol epsilon have been cumbersome due to protease sensitivity and the limited amounts of Pol epsilon in cells. We have developed a protocol for overexpression and purification of Pol epsilon from S. cerevisiae. The native four-subunit complex was purified to homogeneity by conventional chromatography. Pol epsilon was characterized biochemically by sedimentation velocity experiments and gel filtration experiments. The stoichiometry of the four subunits was estimated to be 1:1:1:1 from colloidal Coomassie-stained gels. Based on the sedimentation coefficient (11.9 S) and the Stokes radius (74.5 A), a molecular mass for Pol epsilon of 371 kDa was calculated, in good agreement with the calculated molecular mass of 379 kDa for a heterotetramer. Furthermore, analytical equilibrium ultracentrifugation experiments support the proposed heterotetrameric structure of Pol epsilon. Thus, both DNA polymerase delta and Pol epsilon are purified as monomeric complexes, in agreement with accumulating evidence that Pol delta and Pol epsilon are located on opposite strands of the eukaryotic replication fork.

Links

PubMed Online version:10.1074/jbc.M211818200

Keywords

Catalase; Catalytic Domain; Cell Division; Chromatography; Chromatography, Gel; DNA Polymerase II/chemistry; DNA Polymerase II/metabolism; DNA Polymerase III/chemistry; Dimerization; Electrophoresis, Polyacrylamide Gel; Glycerol/pharmacology; Plasmids/metabolism; Protein Structure, Quaternary; Saccharomyces cerevisiae/enzymology

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

YEAST:DPOE

part_of

GO:0008622: epsilon DNA polymerase complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:DPOE

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P24482

F

Seeded From UniProt

complete

YEAST:DPB2

part_of

GO:0008622: epsilon DNA polymerase complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:DPB2

enables

GO:0005515: protein binding

ECO:0000353: physical interaction evidence used in manual assertion

UniProtKB:P21951

F

Seeded From UniProt

complete

YEAST:DPB3

part_of

GO:0008622: epsilon DNA polymerase complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete

YEAST:DPB4

part_of

GO:0008622: epsilon DNA polymerase complex

ECO:0000314: direct assay evidence used in manual assertion

C

Seeded From UniProt

complete


See also

References

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