DNA Replication in the Gram Positive Bacterium Bacillus Subtilis

该奖项是根据2009年《美国复苏和再投资法案》(公法111-5)提供资金的。到目前为止，大肠杆菌是唯一一个可以从纯化的蛋白质中重组出完整的复制分叉的细胞系统。在这个项目中，将探索一种高度分化的细菌，枯草杆菌，以了解从大肠杆菌模型中建立的哪些原理是通用的，以及哪些变异可以用于染色体的快速、连续、协调复制。枯草杆菌在这方面是一个很有吸引力的系统。通过遗传、分子和细胞生物学的研究，我们知道需要两种不同的复制酶，而不像大肠杆菌系统那样，DNA聚合酶III全酶就足够了。双复制酶系统让人联想到真核系统。人们还知道，与大肠杆菌不同，装载解旋酶需要两种蛋白质，但可能类似于真核生物中可疑的ORC6/CDT1要求。枯草杆菌还使用了两种新的蛋白质，Dna D和Dna B，这两种蛋白质在大肠杆菌中没有同源物。从遗传学上预测的13种蛋白质已经被表达和纯化，并被用来重建一个强大的、滚动的环状复制反应，概括了枯草杆菌染色体在体内复制的一般特征。将进行一项严格的研究，以阐明参与解旋酶组装过程的复制蛋白之间的合作相互作用。DNAE和Polc这两种复制酶的功能将受到干扰，以获得有关其功能的基本信息。该程序的完成将导致对不同模式生物中DNA复制的重要机制的理解。基础知识也将作为较难处理的真核系统中比较研究的原型。该计划将增加本科生和研究生研究人员的培训机会，包括在科学界代表性不足的群体。

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5). To date, E. coli serves as the only cellular system for which a complete replication fork can be reconstituted from purified proteins. In this project, a highly divergent bacterium, Baccilus subtilis, will be explored to learn what principles established from the E. coli model are general and what variations can be employed for the rapid, processive, coordinated replication of a chromosome. B. subtilis is an attractive system in that regard. Through genetic, molecular and cell biology work it is known that two distinct replicases are required, unlike the E. coli system where the DNA polymerase III holoenzyme alone suffices. The two-replicase system is reminiscent of eukaryotic systems. It is also known that two proteins are required to load the helicase, unlike E. coli, but perhaps similar to the suspected Orc6/Cdt1 requirement in eukaryotes. B. subtilis also employs two novel proteins, DnaD and DnaB, that have no homologs in E. coli. Thirteen proteins, predicted from genetics, have been expressed and purified and have been used to reconstitute a robust, rolling circle replicative reaction that recapitulates the general features of B. subtilis chromosomal replication in vivo. A rigorous study will be conducted to elucidate the cooperative interactions of replication proteins that participate in the helicase assembly process. The function of the two replicases, DnaE and PolC, will be perturbed to gain fundamental information regarding their function.Completion of the program will lead to an important mechanistic understanding of DNA replication in a diverse model organism. Fundamental knowledge will also serve as a prototype for comparative studies in less tractable eukaryotic systems. The program will increase training opportunities for undergraduate and graduate researchers, including groups underrepresented in science.