严紧反应下乙酰化修饰对大肠埃希菌DnaA降解调控的研究

When bacteria encounter environmental stresses, for example starvation condition, the stringent response is activated, and DNA replication is halted. Whereas the underlying mechanisms remain unclear. We found that many bacterial proteins were acetylated by proteomics and the acetylation regulated their functions. DnaA controls the initiation of bacterial DNA replication. We found that DnaA was acetylated in Escherichia coli . The acetylation level of DnaA increased and meanwhile the stability of DnaA decreased under carbon starvation. We hypothesized that bacteria could decrease the stability of DnaA by increasing the acetylation level of DnaA under the conditions of carbon or nitrogen starvation. By this mean, cells can promptly decrease the concentration of DnaA and stop DNA replication initiation as well as cell division. Therefore, we will study the relationship between the acetylation of DnaA and the stability of DnaA in vitro and in vivo. We will find whether the acetylation of DnaA regulates the stability of DnaA and how it regulates, especially under the condition of stringent response. Our study will reveal the new regulation mechanism of DNA replication initiation and understand how DNA replication is regulated during the stringent response. The information derived from this project will provide a new perspective for better understanding mechanisms of bacteria’s self-protection against such stresses.

细菌在面对碳源、氮源饥饿等的压力时，会发生严紧反应，导致DNA复制停止，但其调控机制还不完全清楚。我们利用蛋白质组学技术发现细菌中存在广泛的蛋白质乙酰化修饰现象， 其中控制着DNA复制起始的DnaA存在乙酰化修饰，且在碳源饥饿的压力下，胞质DnaA不仅乙酰化水平升高，而且含量显著降低，存在降解现象。我们提出假说：在碳源、氮源饥饿等压力状况下，大肠埃希菌通过调节DnaA的乙酰化修饰来降低DnaA的稳定性，迅速降低胞质DnaA浓度，从而阻止DNA的复制起始，抑制细菌分裂。因此本课题拟采用体内和体外研究策略与方法，探索DnaA乙酰化修饰与其稳定性的关系，研究DNA复制起始调控的机制，深入理解严紧反应过程中DNA的调控，为揭示细菌应对外界压力的自我保护机制提供新的视角。

DNA的复制起始是遗传信息传递和表达的基础，对细菌的生长至关重要。复制起始是细胞周期的中心事件，为了保证复制过程适时有效的进行，细菌体内进化出了多种调控机制。对细菌乙酰化谱的研究结果表明，很多参与复制过程的蛋白都具有乙酰化修饰，但该修饰对复制起始过程的影响却所知甚少。我们前期的研究发现细菌体内的复制起始蛋白DnaA存在乙酰化修饰，且在碳源饥饿的压力下，胞质DnaA不仅乙酰化水平升高，而且含量显著降低，存在降解现象。因此本课题以DnaA的乙酰化修饰为切入点，旨在揭示乙酰化修饰与复制起始功能之间的关系。.在本研究中，结合我们DnaA乙酰化修饰的质谱结果，经质粒互补试验筛选，我们发现DnaA第243位赖氨酸K243是一个关键的乙酰化修饰位点。体内和体外研究表明，K243的乙酰化水平受到乙酰磷酸AcP和CobB的共同调控，并且与胞内AcP浓度正相关。当该位点发生乙酰化修饰时，DnaA蛋白虽然保留与ATP/ADP、dnaA promoter以及DARS DnaA-reactivating sequence, DnaA激活序列的结合能力，但与复制起始位点oriC的结合下降；DNase I足迹实验进一步揭示，该位点发生乙酰化修饰后，DnaA 蛋白不能识别oriC上的一些低亲和力DnaA boxes从而与 oriC 形成一个不完整的起始复合物而无法发挥解链、起始复制的功能。.该研究深入探索了DnaA的乙酰化修饰对DnaA功能的调控作用，揭示了细菌体内新的复制起始调控机制。为研究细菌DNA复制起始的调控作用及蛋白乙酰化修饰的功能提供了新的视角。