Fuctional Analysis of DNA Polymerase ε in Higher Eukaryote

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DNA polymerase ε is essential for cell viability and chromosomal DNA replication in budding yeast. In addition, DNA polymerase ε may be involved in DNA repair and cell-cycle checkpoint control. The enzyme consists of at least four subunits in higher eukaryotic cells as well as in yeast. The largest subunit of DNA polymerase ε is responsible for polymerase activity. To date, the functions of the other subunits have remained unknown. To clarify the functions of DNA polymerase ε by molecular level, the research project was advanced.First of all, the structure of four subunits of mouse DNA polymerase ε was clarified. After growth stimulation by serum addition or UV irradiation, the profiles of the transcriptional level and the translational level for POLE and DPE2 were a little different from those for DPE3 and DPE4, suggesting that all four subunits are not always one complex. The interaction between POLE and DPE2 contributed to the stabilization of polymerase activity.With a view to elucidating the functions of the second largest subunit of mouse DNA polymerase ε (DPE2), yeast two-hybrid screening was performed to identify mouse proteins that interact with this subunit. SAP18, a polypeptide associated with co-repressor protein Sin3, was identified as an interacting protein. The interaction induced repression of transcription in reporter plasmid assays, which was inhibited by trichostatin A. Moreover, POLE directly interacts with Sin3 through the results of immuno-precipitation experiment. These results indicate that DPE2 may recruit histone deacetylase (HDAC) to the replication fork to modify the chromatin structure.