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Structure of eukaryotic DNA polymerase epsilon and lesion bypass capability
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
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 [en]
DNA polymerase epsilon, DNA replication, Okazaki fragment, Translesion synthesis, DNA lesion, dNTP
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-1477ISBN: 978-91-7264-434-2 (print)OAI: oai:DiVA.org:umu-1477DiVA: diva2:141190
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
List of papers
1. Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication.
Open this publication in new window or tab >>Idling by DNA polymerase delta maintains a ligatable nick during lagging-strand DNA replication.
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2004 (English)In: Genes & Development, ISSN 0890-9369, Vol. 18, no 22, 2764-2773 p.Article in journal (Refereed) Published
Keyword
DNA/*metabolism, DNA Helicases/metabolism, DNA Polymerase III/*metabolism, DNA Primers, DNA Replication, Exonucleases/metabolism, Flap Endonucleases/*metabolism, Oligonucleotides/metabolism, Saccharomyces cerevisiae/*enzymology/*genetics
Identifiers
urn:nbn:se:umu:diva-6352 (URN)10.1101/gad.1252304 (DOI)15520275 (PubMedID)
Available from: 2007-12-09 Created: 2007-12-09Bibliographically 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, 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: 2007-12-09Bibliographically approved
3. DNA polymerase epsilon bypasses an abasic site in the absence of a processivity clamp
Open this publication in new window or tab >>DNA polymerase epsilon bypasses an abasic site in the absence of a processivity clamp
Manuscript (Other academic)
Identifiers
urn:nbn:se:umu:diva-2874 (URN)
Available from: 2008-01-07 Created: 2008-01-07 Last updated: 2010-01-13Bibliographically approved
4. Evidence for lesion bypass by yeast replicative DNA polymerases during DNA damage
Open this publication in new window or tab >>Evidence for lesion bypass by yeast replicative DNA polymerases during DNA damage
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2008 (English)In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 36, no 17, 5660-5667 p.Article in journal (Refereed) Published
Abstract [en]

The enzyme ribonucleotide reductase, responsible for the synthesis of deoxyribonucleotides (dNTP), is upregulated in response to DNA damage in all organisms. In Saccharomyces cerevisiae, dNTP concentration increases approximately 6- to 8-fold in response to DNA damage. This concentration increase is associated with improved tolerance of DNA damage, suggesting that translesion DNA synthesis is more efficient at elevated dNTP concentration. Here we show that in a yeast strain with all specialized translesion DNA polymerases deleted, 4-nitroquinoline oxide (4-NQO) treatment increases mutation frequency approximately 3-fold, and that an increase in dNTP concentration significantly improves the tolerance of this strain to 4-NQO induced damage. In vitro, under single-hit conditions, the replicative DNA polymerase epsilon does not bypass 7,8-dihydro-8-oxoguanine lesion (8-oxoG, one of the lesions produced by 4-NQO) at S-phase dNTP concentration, but does bypass the same lesion with 19-27% efficiency at DNA-damage-state dNTP concentration. The nucleotide inserted opposite 8-oxoG is dATP. We propose that during DNA damage in S. cerevisiae increased dNTP concentration allows replicative DNA polymerases to bypass certain DNA lesions.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:umu:diva-22414 (URN)10.1093/nar/gkn555 (DOI)18772226 (PubMedID)
Available from: 2009-05-07 Created: 2009-05-07 Last updated: 2017-12-13Bibliographically approved

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