Fuctional Analysis of DNA Polymerase ε in Higher Eukaryote

DNA聚合酶ε对出芽酵母的细胞活力和染色体DNA复制至关重要。此外，DNA聚合酶ε可能参与DNA修复和细胞周期检查点控制。在高等真核细胞和酵母中，这种酶至少由四个亚基组成。DNA聚合酶ε的最大亚基负责聚合酶的活性。迄今为止，其他亚单位的功能仍然未知。为了从分子水平上阐明DNA聚合酶ε的功能，提出了研究方向。首先，明确了小鼠DNA聚合酶ε的4个亚基的结构。在血清添加或紫外线照射刺激生长后，POLE和DPE2的转录水平和翻译水平与DPE3和DPE4略有不同，这表明这四个亚基并不总是一个复合体。POLE和DPE2之间的相互作用有助于稳定聚合酶的活性。为了阐明小鼠DNA聚合酶ε (DPE2)第二大亚基的功能，采用酵母双杂交筛选方法鉴定了与该亚基相互作用的小鼠蛋白。SAP18是一种与协同抑制蛋白Sin3相关的多肽，是一种相互作用的蛋白。在报告质粒实验中，这种相互作用诱导了转录抑制，而这种抑制被曲古斯汀a抑制。此外，通过免疫沉淀实验的结果，POLE直接与Sin3相互作用。这些结果表明，DPE2可能在复制叉上募集组蛋白去乙酰化酶（HDAC）来修饰染色质结构。

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.