DNA polymerase ε : structure of the human and mouse genes for the catalytic A-subunit, transcriptional regulation of the human gene for the B-subunit, and identification of DNA topoisomerase IIβ binding protein as a partner of DNA polymerase ε
1University of Oulu, Biocenter Oulu
2University of Oulu, Faculty of Science, Department of Biochemistry
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|Academic Dissertation to be presented with the assent of the Faculty of Science, University of Oulu, for public discussion in Kuusamonsali (Auditorium YB 210), Linnanmaa, on December 8th, 2000, at 12 noon.
Professor Leena Alhonen
Professor Tuomas Sopanen
The human and mouse genes POLE1 and Pole1 for the catalytic subunit of DNA polymerase ε contain 51 and 49 exons, respectively, and the human gene POLE2 for the B-subunit contains 19 exons. The human POLE1 encodes three alternatively spliced mRNAs differing in their 5'-terminal sequence and in the N-termini of the predicted proteins. The promoters for the major human transcript and the mouse Pole1 are G+C rich, TATA-less and contain putative cis-acting elements typical of both S phase upregulated and serum responsive promoters. Interestingly, the three human alternative transcripts are expressed from three promoters, and other structural features of POLE1 suggest that regulation of its expression is complicated. The amino acid sequence of the catalytic subunit deduced from the mouse cDNA shows remarkable evolutionary conservation in the DNA polymerase ε family. Interestingly, several conserved elements involved in template-primer binding differ from those of other class B DNA polymerases. This is likely to reflect a distinctive function of the enzyme. The mouse Pole1 was localized to chromosome 5 region E3-E5.
The expression of the human POLE2 encoding the B-subunit of DNA polymerase ε is dependent on cell proliferation in a late serum-responsive manner. This is typical for DNA replication-related proteins. The promoter, which utilizes multiple transcriptional initiation sites, is G+C rich and lacks a TATA-box. A 75 bp core promoter region is located within exon 1 and contains an Sp1 element as a critical determinant of the promoter activity. Two overlapping E2F elements adjacent to the Sp1 element are essential for full promoter activity and serum response. Immediately downstream from the core promoter region reside binding sites for E2F1 and NF-1. POLE2 seems to be regulated by two E2F-pocket protein complexes, one associated with Sp1 and the other with NF-1.
The complete cDNA of the human DNA topoisomerase IIβ binding protein (TopBP1) reveals a 170 kDa protein that contains eight BRCT-domains and shows homology to S. cerevisiae Dpb11 and S. pombe Cut5/Rad4. The protein interacts physically with human DNA polymerase ε as shown by co-immunoprecipitation. A peptide containing the 6th BRCT-domain and an antibody against this peptide inhibit DNA replication in isolated nuclei, indicating that the protein is required for DNA replication. The expression of TopBP1 is proliferation-dependent in a manner that is typical for replication proteins. The gene encoding TopBP1 was localized to chromosome 3q21-q23.
Acta Universitatis Ouluensis. D, Medica
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