DNA Replication in B. subtilis

描述（由申请人提供）： 几十年的研究为我们提供了关于大肠杆菌中DNA复制的详细信息。细菌基因组序列的爆炸性增长使人们能够在标准的E.线圈原型这些研究的目的是建立一个完整的复制系统的第二个非常遥远的细菌，B。枯草杆菌。B。枯草芽孢杆菌理想地适合于这些目的，因为i.它是多样的，属于低GC革兰氏（+）分类，进化遥远（>> 10亿年）。从变形菌纲（Proteobacteria）和E. coli，ii.其基因组已被测序，并具有发达的遗传系统，iii.它可以在发酵罐中大量生长，使其适合于生化分析，iv）基因组学表明，从DNA复制的角度来看，它代表了最多样化的细菌群（具有两种明显不同的复制酶和许多复制蛋白，而在大肠杆菌中没有对应物）。大肠杆菌，和v）它提供了代表一组重要的人类病原体（链球菌、葡萄球菌和肠球菌）的模型系统。建议资助期内的研究，是建基于未有拨款的初步研究所建立的稳固基础，并会集中于建立一个独立的研究机构。伸长装置部件和功能的特性，ii.提供RNA引物所需的蛋白质，和iii.这些蛋白质是起源特异性启动所需的。重点将放在这些特点的B。subtilis的复制系统，使其不同于E.杆菌这将提供关于细菌复制系统之间在基本组成部分、机制和调节方面可以预期的变化的重要信息。了解含有两种DNA聚合酶Ills的生物体中DNA复制的生化细节，可以提供适用于其他两种聚合酶复制酶的范式，包括真核生物，其中Pol epsilon和delta似乎都参与染色体的进行性复制。

DESCRIPTION (provided by applicant): Decades of study have provided us with detailed information about DNA replication in E.coli. The explosion of bacterial genomic sequences has enabled facile exploration beyond the standard E. coil prototype. The objective of these proposed studies is to establish a complete replication system for a second very distant bacterium, B. subtilis. B. subtilis is ideally suited for these purposes because i. it is diverse, belonging to the low GC Gram (+) classification, evolutionarily distant (>>1 billion yr.) from Proteobacteria and E. coli, ii. its genome has been sequenced and it has a developed genetic system, iii. it can be grown in fermentors in large quantity making it suitable for biochemical analysis, iv) genomics indicates it is representative of the most diverse group of bacteria from a DNA replication standpoint (having two apparent distinct replicases and a number of replication proteins with no counterpart in E. coli, and v) it provides a model system representative of an important group of important human pathogens (streptococci, staphylococci and enterococci. The studies for the proposed grant period build upon a strong base established through unfunded preliminary studies and will focus upon establishing i. the identity of the elongation apparatus components and function, ii. the proteins required for providing RNA primers and iii. the proteins required for origin-specific initiation. Emphasis will be placed on those features of the B. subtilis replication system that make it distinct from E. coli. This will provide important information regarding the variations that can be expected between bacterial replication systems in terms of basic components, mechanisms and regulation. Understanding the biochemical details of DNA replication in an organism that contains two DNA polymerase Ills, may provide paradigms applicable to other two-polymerase replicases including eukaryotes where Pol epsilon and delta both appear to be involved in processive chromosomal replication.