Roles of DNA polymerase epsilon and TopBP1 in DNA replication and damage response
1University of Oulu, Faculty of Science, Department of Biochemistry
2University of Oulu, Biocenter Oulu
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|Academic dissertation to be presented, with the assent of the Faculty of Science of the University of Oulu, for public defence in Raahensali (Auditorium L10), Linnanmaa, on December 15th, 2006, at 12 noon
Docent Varpu Marjomäki
Docent Minna Nyström
During DNA replication cells accurately copy their DNA to transfer the genetic information to daughter cells. DNA polymerases synthesise the new DNA strand using the old strand as a template. Other functions of DNA polymerases are recombination linked and DNA iamage repair linked DNA synthesis, regulation of replication complex formation and regulation of transcription — a process in which the genetic information is transformed into an RNA sequence needed to guide protein synthesis.
In this study, the TopBP1 protein was shown to associate with DNA polymerase epsilon. TopBP1 contains eight BRCT domains mediating interactions between phosphorylated proteins and is a human homolog of bakers yeast Dpb11 and fission yeast Cut5. These yeast proteins act on DNA replication and cell cycle arrest after DNA damage. TopBP1 was found to be phosphorylated and expressed in elevated amounts during S phase suggesting an involvement in DNA replication. This was directly demonstrated by DNA synthesis inhibition by a competing TopBP1 fragment and by an antibody targeted to block TopBP1.
Ultraviolet irradiation damages DNA and decreases the amount of TopBP1 in the nucleus. The transcription factor Miz-1 was found to associate with TopBP1 and was released from this interaction after UV damage. Free Miz-1 activated the expression of the cell cycle arresting proteins p15 and p21 cooperatively with other transcription factors and allowed extra time for DNA damage repair.
TopBP1 was also found to interact with the breast cancer susceptibility protein 1 and both proteins localised together to arrested DNA synthesis apparatuses. The interaction of TopBP1 with the damage recognition and processing protein Rad9 is still further evidence of a link between TopBP1 and DNA damage.
DNA polymerase epsilon forms a complex with Cdc45, a protein involved in DNA replication initiation and elongation. This complex does not interact with Cdc45 complexed with DNA polymerase delta, suggesting that these complexes synthesise DNA independently of each other. Our results are in agreement with the view that polymerase epsilon synthesises the first strand of DNA and polymerase delta the other.
Finally,DNA polymerase epsilon binds to the RNA synthesising form of RNA polymerase II and nascent transcripts. The physiological meaning of this interaction needs to be determined.
Acta Universitatis Ouluensis. A, Scientiae rerum naturalium
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