酵母RNaseH2磷酸化调控机制及其生理功能的研究

The eukaryotic RNase H2 has an important role in DNA replication, repair and transcription. Some mutation of human RNase H2 gene can lead to Aicardi Goutie`res syndrome (AGS) syndrome by disturbing protein post-translational modification. However, the regulatory mechanism of post-translational modification of RNase H2 is still not clear, which greatly hinders the study of RNase H2 and AGS pathogenesis. We intend to use well-studied eukaryotic organism, S.cerevisiae, to study phosphorylation regulatory mechanism of RNase H2. The research includes the determination of phosphorylation of yeast RNase H2 by the Bud32, Cak1p or Kss1 in vitro and vivo respectively, as well as the phosphorylation sites of RNase H2. Moreover, in order to study the function of RNase H2, the protein stability,enzymatic activity, the interaction of other proteins with RNase H2 and their effect on RNase H2 enzymatic activity will be determined with yeast RNase H2 being phosphorylated or not respectively. Based on the results mentioned above, we will explain the phosphorylation regulatory mechanism of yeast RNase H2 induced by Bud32p, Cak1p or Kss1p, and the influence of phosphorylation on the structure and function of RNase H2. The role of phosphorylated RNase H2 on DNA replication and repair will also be speculated. We believed that our work will promote the study of the pathogenesis of human AGS syndrome.

真核生物RNaseH2在生物体DNA复制、修复和RNA转录等方面具有重要作用。一些人类RNaseH2基因突变可破坏RNaseH2蛋白的翻译后修饰，引起AGS综合征。然而RNaseH2翻译后修饰的调控机制仍然并不清楚，这极大阻碍了RNaseH2胞内功能和AGS发病机理的研究。本项目拟以模式生物酵母为例，研究RNaseH2磷酸化调控机制。具体研究内容包括细胞内外测定Bud32、Cak1p或Kss1蛋白对RNaseH2的磷酸化作用，确定磷酸化位点；测定磷酸化修饰前后 RNaseH2蛋白稳定性、酶活性和其他蛋白与RNaseH2的相互作用以及这些蛋白对RNaseH2酶促反应的影响。根据以上结果阐明Bud32p、Cak1p或Kss1p蛋白磷酸化调控酵母RNaseH2的作用机制以及磷酸化对RNaseH2结构和功能的影响，推测磷酸化RNaseH2影响DNA复制或修复的机制，促进AGS综合征发病机制的研究。

真核生物RNaseH2在生物体DNA复制、修复和RNA转录等方面具有重要作用。RNaseH2的磷酸化调控机制对RNaseH2胞内功能以及AGS发病机理的研究具有重要的理论和应用价值。本研究利用建立的蛋白质组定性和定量分析平台，以模式生物酵母为例，详细分析了Bud32蛋白与RNaseH2复合物的相互作用以及Bud32对RNaseH2的磷酸化作用，鉴定磷酸化作用位点。另外，测定磷酸化修饰前后RNaseH2稳定性、酶活性和其他蛋白与RNaseH2的相互作用以及这些蛋白对RNaseH2酶促反应的影响。本项目的研究成果将有利于酵母RNaseH2的磷酸化调控作用机制以及磷酸化RNaseH2结构和功能的研究。