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Structure of Saccharomyces cerevisiae DNA polymerase epsilon by cryo-electron microscopy.
Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
Umeå University, Faculty of Medicine, Medical Biochemistry and Biophsyics.
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2006 (English)In: Nature Structural & Molecular Biology, ISSN 1545-9993, Vol. 13, no 1, 35-43 p.Article in journal (Refereed) Published
Place, publisher, year, edition, pages
2006. Vol. 13, no 1, 35-43 p.
Keyword [en]
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: urn:nbn:se:umu:diva-6351DOI: 10.1038/nsmb1040PubMedID: 16369485OAI: oai:DiVA.org:umu-6351DiVA: diva2:146020
Available from: 2007-12-09 Created: 2007-12-09Bibliographically approved
In thesis
1. Structure of eukaryotic DNA polymerase epsilon and lesion bypass capability
Open this publication in new window or tab >>Structure of eukaryotic DNA polymerase epsilon and lesion bypass capability
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

To transfer the information in the genome from mother cell to daughter cell, the DNA replication must be carried out only once and with very high fidelity prior to every cell division. In yeast there are several different DNA polymerases involved in DNA replication and/or DNA repair. The two replicative DNA polymerases, DNA polymerase delta (Pol delta) and DNA polymerase epsilon (Pol epsilon), which both include a proofreading 3´→5´exonuclease activity, can replicate and proofread the genome with a very high degree of accuracy. The aim of this thesis was to gain a better understanding of how the enigmatic DNA polymerase epsilon participates in DNA transactions.

To investigate whether Pol epsilon or Pol delta is responsible for the synthesis of DNA on the lagging strand, the processing and assembly of Okazaki fragments was studied. Pol delta was found to have a unique property called “idling” which, together with the flap-endonuclease (FEN1), maintained a ligatable nick for DNA ligase I. In contrast, Pol epsilon was found to lack the ability to “idle” and interact functionally with FEN-1, indicating that Pol epsilon is not involved in processing Okazaki fragments. Together with previous genetic studies, it was concluded that Pol delta is the preferred lagging strand polymerase, leaving Pol epsilon to carry out some other function.

The structure of Pol epsilon was determined by cryo-electron microscopy, to a resolution of ~20 Å. Pol epsilon is composed of a globular “head” domain consisting of the large catalytic subunit Pol2p, and a “tail” domain, consisting of the small subunits Dpb2p, Dpb3p, and Dpb4p. The two separable domains were found to be connected by a flexible hinge. Interestingly, the high intrinsic processivity of Pol epsilon depends on the interaction between the tail domain and double-stranded DNA.

As a replicative DNA polymerase, Pol epsilon encounters different lesions in DNA. It was shown that Pol epsilon can perform translesion synthesis (TLS) through a model abasic site in the absence of external processivity clamps under single-hit conditions. The lesion bypass was dependent of the sequence on the template and also on a proper interaction of the “tail”domain with the primer-template.

Yeast cells treated with a DNA damaging agent and devoid of all TLS polymerases showed improved survival rates in the presence of elevated levels of dNTPs. These genetic results suggested that replicative polymerases may be engaged in the bypass of some DNA lesions. In vitro, Pol epsilon was found to bypass 8-OxoG at elevated dNTP levels. Together, the in vitro and in vivo results suggest that the replicative polymerases may be engaged in bypass of less bulky DNA lesions at elevated dNTP levels.

In conclusion, the low-resolution structure presented represents the first structural characterization of a eukaryotic multi-subunit DNA polymerase. The replicative DNA polymerase Pol epsilon can perform translesion synthesis due to an interaction between the tail domain and double-stranded DNA. Pol epsilon may also bypass less bulky DNA lesions when there are elevated dNTP concentrations in vivo.

Place, publisher, year, edition, pages
Umeå: Medicinsk kemi och biofysik, 2008. 42 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1136
Keyword
DNA polymerase epsilon, DNA replication, Okazaki fragment, Translesion synthesis, DNA lesion, dNTP
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-1477 (URN)978-91-7264-434-2 (ISBN)
Public defence
2008-01-25, KB3A9, KBC, Umeå Universitet, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2008-01-07 Created: 2008-01-07 Last updated: 2010-08-23Bibliographically approved
2. Functional and structural properties of eukaryotic DNA polymerase epsilon
Open this publication in new window or tab >>Functional and structural properties of eukaryotic DNA polymerase epsilon
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
eukaryotic DNA replication, DNA polymerase epsilon, protein-DNA interaction
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-837 (URN)91-7264-131-2 (ISBN)
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

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Sabouri, NasimJohansson, Erik

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