University of Oulu

The role of DNA polymerases, in particular DNA polymerase ε in DNA repair and replication

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Author: Pospiech, Helmut
Organizations: University of Oulu, Biocenter Oulu
University of Oulu, Faculty of Science, Department of Biochemistry
Format: eBook
Online Access: PDF Full Text (PDF, 1.1 MB)
Persistent link: http://urn.fi/urn:isbn:9514266692
Language: English
Published: 2002
Publish Date: 2002-04-19
Thesis type: Doctoral Dissertation
Defence Note: Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Kajaaninsali (Auditorium L6), Linnanmaa, on April 19th, 2002, at 10 a.m.
Reviewer: Docent Minna Nyström-Lahti
Professor Olli Silvennoinen
Description:

Abstract

Analysis of the primary structure of DNA polymerase ε B subunit defined similarities to B subunits of eukaryotic DNA polymerases α, δ and ε as well as the small subunits of DNA polymerase DI of Euryarchaeota. Multiple sequence alignment of these proteins revealed the presence of 12 conserved motifs and defined a novel protein superfamily. The members of the B subunit family share a common domain architecture, suggesting a similar fold, and arguing for a conserved function among these proteins.

The contribution of human DNA polymerase ε to nuclear DNA replication was studied using the antibody K18 that specifically inhibits the activity of this enzyme in vitro. This antibody significantly inhibited DNA synthesis both when microinjected into nuclei of exponentially growing human fibroblasts and in isolated HeLa cell nuclei, but did not inhibit SV40 DNA replication in vitro. These results suggest that the human DNA polymerase ε contributes substantially to the replicative synthesis of DNA and emphasises the differences between cellular replication and viral model systems.

The human DNA polymerases ε and δ were found capable of gap-filling DNA synthesis during nucleotide excision repair in vitro. Both enzymes required PCNA and the clamp loader RFC, and in addition, polymerase δ required Fen-1 to prevent excessive displacement synthesis. Nucleotide excision repair of a defined DNA lesion was completely reconstituted utilising largely recombinant proteins, only ligase I and DNA polymerases δ and ε provided as highly purified human enzymes. This system was also utilised to study the role of the transcription factor II H during repair.

Human non-homologous end joining of model substrates with different DNA end configurations was studied in HeLa cell extracts. This process depended partially on DNA synthesis as an aphidicolin-dependent DNA polymerase was required for the formation of a subset of end joining products. Experiments with neutralising antibodies reveal that DNA polymerase α but not DNA polymerases β or ε, may represent this DNA polymerase activity. Our results indicate that DNA synthesis contributes to the stability of DNA ends, and influences both the efficiency and outcome of the end joining event. Furthermore, our results suggest a minor role of PCNA in non-homologous end joining.


Series: Acta Universitatis Ouluensis. A, Scientiae rerum naturalium
ISSN-E: 1796-220X
ISBN: 951-42-6669-2
ISBN Print: 951-42-6668-4
Issue: 381
Subjects:
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