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.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:isbn951-42-6669-2 |
| Date | 19 April 2002 |
| Creators | Pospiech, H. (Helmut) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © University of Oulu, 2002 |
| Relation | info:eu-repo/semantics/altIdentifier/pissn/0355-3191, info:eu-repo/semantics/altIdentifier/eissn/1796-220X |
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