Fkh家族转录因子在酿酒酵母DNA复制时序调控中的作用机理

DNA replication ensures the accurate duplication and transmition of genetic information in all types of cells. Eukaryotic DNA replication initiates from hundreds to thousands of origins which is highly coordinated in a spatial-temporal manner during S-phase of the cell cycle. DNA replication timing is conserved in eukaryotic organisms, the dysregulation of DNA replication timing correlates with genome instability and tumorigenesis. In Saccharomyces cerevisiae, DDK(Dbf4-dependent kinase) is involved in MCM2-7 helicase activation during DNA replication initiation. However, how Dbf4 is preferentially recruited to early origins remains obscue. In a unpublished high throughput yeast two hybrid screen, we have identified three Dbf4 interactors, transcription factors Fkh1/Fkh2 and another essential component of replication initiation complex Sld3. Such physical associations were further confirmed by coimmunoprecipitation. Moreover, when Fkh1 and Fkh2 were deleted, enrichment of Dbf4 at early origins was significantly decreased and the firing of early origins was also delayed. Here, we plan to characterize the details of these new interactions between Fkh and DDK, ellucidate the role of Fkh in recruitment of DDK to early origins. We’ll investigate the role and molecular mechanism of Fkh in control of DNA replication timing, which will inevitably contribute to understanding the spatio-temporal control of DNA replication itself and coordination with other important DNA activities such as transcription and chromatin dynamics.

真核生物基因组DNA复制起始于数百到数万个复制原点。这些原点并不同步启动，而是呈现一定的先后顺序。酿酒酵母的复制原点分为明显的“早”、“中”、“晚”三类，是研究真核DNA复制时序调控的理想模型。现有的“限量因子模型”认为，DNA复制时序是由Dbf4-Cdc7 （DDK）蛋白激酶等限量因子决定的。但限量因子到底是如何优先被募集到早期复制原点这一关键问题尚不明确。本课题组前期通过高通量酵母双杂交筛选到DDK的新的互作蛋白，转录因子Fkh1/Fkh2。ChIP实验初步证明Dbf4在早期原点的募集依赖Fkh1。本项目拟在此基础上，进一步解析Fkh-DDK互作的分子细节，揭示Fkh转录因子在DDK等限量因子招募中的作用及其分子机制，探讨真核生物染色体着丝粒和其它区域DNA复制时序控制机制的异同点。本研究将加深对DNA复制时空调控、尤其是与转录等其它DNA代谢过程协调等生命科学基本课题的认识。

真核生物基因组DNA复制起始原点的启动呈现一定的先后顺序。现有的“限量因子模型”认为DNA复制时序是由Dbf4-Cdc7（DDK）等限量因子决定的，但限量因子如何被优先招募到早期复制原点的机制并不明确。本项目以酿酒酵母为模式生物，通过遗传和生化等手段，发现转录因子Fkh1/Fkh2在DNA复制时序调控过程中促进DDK在早期复制原点招募的分子机制；DDK激酶通过磷酸化Mcm2和Mcm6的N端促进限量因子Sld3的招募，从而介导CMG复合体的装配，激活复制原点。而当遇到复制压力时，Mck1和Dun1对dNTPs合成的限速酶——核糖核苷酸还原酶 (Ribonucleotide Reductase, RNR) 的表达具有明确分工，Dun1主要负责早期响应, 而Mck1主要负责晚期响应，从而保证细胞内dNTPs水平维持在一个适当并且平衡的水平。本项目加深了对DNA复制时空调控，尤其是与转录等其他DNA代谢过程协调过程的认识。