真核细胞染色体DNA复制机理研究

Chromosomal DNA replication is a central biological event. To elucidate the mechanism of this event is of paramount importance. Although a great progress has been made in understanding how DNA is replicated in eukaryotes, many critical questions still remain to be answered. In this proposal, we plan to study some of those long-standing questions. How is the function of replication initiation protein Sap1 regulated? How are human DNA replication origins genome-widely distributed and selected? How does the DNA replication initiation protein Girdin (the homologue of Sap1 in metazoans) interact with DNA? Does human ORC have an accessory protein that is responsible for ORC to bind to DNA? How do DNA replication forks traverse through nucleosomes or chromatin structures? All these questions require to be answered in order to have a deep understanding of eukaryotic DNA replication event that is extremely complex and sophisticatedly regulated.

染色体DNA复制是一个核心生物事件，阐明其机制具有极其重要性。虽然真核细胞DNA复制的机理研究已有重要进展，但仍有许多重要问题等待解决。在这个研究计划，我们计划研究几个长期悬而未决的问题。DNA复制起始蛋白Sap1的功能是如何被调控的？人细胞DNA复制源是如何在全基因组范围内安排的？及他们是如何被选择的？DNA复制起始蛋白Girdin如何与DNA相互作用？人细胞ORC有一个附属蛋白吗？是这个附属蛋白把ORC带到DNA复制源吗？DNA复制叉是如何通过核小体或染色质结构的？为对真核细胞DNA复制机理有一个深刻的理解，这些问题亟待解决。

染色体DNA复制是一个核心生物事件，阐明其机制具有极其重要性。虽然真核细胞DNA 复制的机理研究已有重要进展，但仍有许多重要问题等待解决。通过本项目的研究，我们阐明了复制起始蛋白Sap1的调控机制, 发现Sap1的N-端的2个丝氨酸被CDK磷酸化，导致其生物功能减弱，防止一个细胞周期内DNA复制的重复起始；证明Girdin是DNA复制起始蛋白，直接参与pre-RC组装；ChIP-seq实验确定人细胞DNA复制源，并阐明其序列特征及结构；发现新的ORC相互作用蛋白，并证明该蛋白是高等真核细胞ORC的必须组成成分，是DNA复制的必需蛋白，及介导ORC与DNA的相互作用；发现调控维持停顿复制叉稳定性的新通路，并命名为chromsfork control。