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Functional and structural properties of eukaryotic DNA polymerase epsilon
Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
2006 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In eukaryotes there are three DNA polymerases which are essential for the replication of chromosomal DNA: DNA polymerase alpha (Pol alpha), DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon). In vitro studies of viral DNA replication showed that Pol alpha and Pol delta are sufficient for DNA replication on both leading and lagging DNA strands, thus leaving the function of Pol epsilon unknown. The low abundance and the reported protease sensitivity of Pol epsilon were holding back biochemical studies of the enzyme. The aim of this study was to characterize the structural and functional properties of eukaryotic Pol epsilon.

We first developed a protocol for over-expression and purification of Pol epsilon from the yeast Saccharomyces cerevisiae. Pol epsilon consists of four subunits: Pol2 (catalytic subunit), Dpb2, Dpb3 and Dpb4. This four-subunit complex was purified to homogeneity by conventional chromatography and the subunit stoichiometry of purified Pol epsilon was estimated from colloidal coomassie-stained gels to be 1:1:1:1. The quaternary structure was determined by sedimentation velocity and gel filtration experiments. Molecular mass (371 kDa) was calculated from the experimentally determined Stokes radius (74.5 Å) and sedimentation coefficient (11.9 S) and was in good agreement with a theoretical molecular mass calculated for a heterotetramer (379 kDa). Analytical sedimentation equilibrium ultracentrifugation experiments supported the proposed heterotetrameric structure of Pol epsilon.

By cryo-electron microscopy and single-particle image analysis we determined the structure of Saccharomyces cerevisiae Pol epsilon to 20-Å resolution. The four-subunit complex was found to consist of a globular domain, comprising the Pol2 subunit, flexibly connected to an elongated domain, including Dpb2, Dpb3 and Dpb4 subunits. We found that Pol epsilon requires a minimal length of 40 base pairs of primer-template duplex to be processive. This length corresponds to the dimensions of the elongated domain.

To characterize the fidelity by which Pol epsilon synthesizes DNA, we purified wild type and exonuclease-deficient Pol epsilon. Wild type Pol epsilon synthesizes DNA with a very high accuracy. Analysis of the exonuclease-deficient Pol epsilon showed that Pol epsilon proofreads more than 90% of the errors made by its polymerase activity. Exonuclease-deficient Pol epsilon was shown to have a specific spectrum of errors not seen in other DNA polymerases: a high proportion of transversions resulting from T-dTTP, T-dCTP and C-dTTP mispairs. This unique error specificity and amino acid sequence alignment suggest that the structure of the polymerase active site of Pol epsilon differs from those of other members of B family DNA polymerases.

With recombinant proteins and circular single-stranded DNA templates, we partially reconstituted DNA replication in vitro, in which we challenged Pol epsilon and Pol delta in side-by-side comparisons regarding functional assays for polymerase activity and processivity, as well as physical interactions with nucleic acids and PCNA. We found that Pol epsilon activity and “on-DNA” PCNA interactions are dependent on RPA-coated template DNA. By the surface plasmon resonance technique, we showed that Pol epsilon has a high affinity for DNA and low affinity for immobilized PCNA. By contrast, Pol delta was found to have low affinity for DNA and high affinity for PCNA. We suggest that a possible function of RPA is to regulate down the DNA synthesis through Pol epsilon, and that the mechanism by which Pol epsilon and Pol delta load onto the template is different due to different properties of the interaction with DNA and PCNA.

Place, publisher, year, edition, pages
Umeå: Medicinsk biokemi och biofysik , 2006. , 38 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 0346-6612
Keyword [en]
eukaryotic DNA replication, DNA polymerase epsilon, protein-DNA interaction
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-837ISBN: 91-7264-131-2 (print)OAI: oai:DiVA.org:umu-837DiVA: diva2:144678
Public defence
2006-09-22, KB3A9, KBC-huset, Umeå university, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2006-08-30 Created: 2006-08-30 Last updated: 2009-09-30Bibliographically approved
List of papers
1. The quaternary structure of DNA polymerase epsilon from Saccharomyces cerevisiae.
Open this publication in new window or tab >>The quaternary structure of DNA polymerase epsilon from Saccharomyces cerevisiae.
2003 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 16, 14082-14086 p.Article in journal (Refereed) Published
Keyword
Catalase, Catalytic Domain, Cell Division, Chromatography, Chromatography; Gel, DNA Polymerase II/*chemistry/metabolism, DNA Polymerase III/chemistry, Dimerization, Electrophoresis; Polyacrylamide Gel, Glycerol/pharmacology, Plasmids/metabolism, Protein Structure; Quaternary, Saccharomyces cerevisiae/*enzymology
Identifiers
urn:nbn:se:umu:diva-10279 (URN)10.1074/jbc.M211818200 (DOI)12571237 (PubMedID)
Available from: 2008-08-11 Created: 2008-08-11 Last updated: 2017-12-14Bibliographically approved
2. Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron microscopy.
Open this publication in new window or tab >>Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron microscopy.
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2006 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, E-ISSN 1545-9985, Vol. 13, no 1, 35-43 p.Article in journal (Refereed) Published
Keyword
Catalysis, Cryoelectron Microscopy, DEAD-box RNA Helicases, DNA Polymerase II/*chemistry/metabolism/*ultrastructure, DNA; Fungal/chemistry/metabolism/ultrastructure, Models; Molecular, Protein Binding, Protein Structure; Quaternary, Protein Structure; Tertiary, Protein Subunits/chemistry/metabolism, RNA Helicases/chemistry/metabolism/ultrastructure, Saccharomyces cerevisiae/*enzymology, Saccharomyces cerevisiae Proteins/chemistry/metabolism/ultrastructure
Identifiers
urn:nbn:se:umu:diva-6351 (URN)10.1038/nsmb1040 (DOI)16369485 (PubMedID)
Available from: 2007-12-09 Created: 2007-12-09 Last updated: 2017-12-14Bibliographically approved
3. Unique error signature of the four-subunit yeast DNA polymerase epsilon.
Open this publication in new window or tab >>Unique error signature of the four-subunit yeast DNA polymerase epsilon.
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2003 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 278, no 44, 43770-43780 p.Article in journal (Refereed) Published
Keyword
Amino Acid Sequence, Base Pair Mismatch, Base Sequence, DNA Mutational Analysis, DNA Polymerase II/*chemistry, DNA Repair, DNA Replication, DNA-Directed DNA Polymerase/metabolism, Electrophoresis; Polyacrylamide Gel, Exonucleases/metabolism, Frameshift Mutation, Models; Molecular, Molecular Sequence Data, Mutation, Phenotype, Saccharomyces cerevisiae/*enzymology, Sequence Homology; Amino Acid
Identifiers
urn:nbn:se:umu:diva-10278 (URN)10.1074/jbc.M306893200 (DOI)12882968 (PubMedID)
Available from: 2008-08-11 Created: 2008-08-11 Last updated: 2017-12-14Bibliographically approved
4. Similarities and differences between the two replicative DNA polymerases, DNA polymerase delta and DNA polymerase epsilon
Open this publication in new window or tab >>Similarities and differences between the two replicative DNA polymerases, DNA polymerase delta and DNA polymerase epsilon
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Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-5228 (URN)
Available from: 2006-08-30 Created: 2006-08-30 Last updated: 2010-01-13Bibliographically approved

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