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有关大肠杆菌中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.线圈原型之外进行便捷的探索。这些拟议研究的目标是为第二种非常遥远的细菌枯草杆菌建立一个完整的复制系统。枯草杆菌非常适合于这些目的，因为I。它是多样化的，属于低GC Gram(+)分类，进化上很遥远(&gt；&gt；10亿年。)来自变形杆菌和大肠杆菌，II。它的基因组已经测序，它有一个发达的遗传系统，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体(具有两个明显的不同复制酶和一些复制蛋白，而在大肠杆菌中没有对应的复制蛋白)；v)它提供了一个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。拟议赠款期间的研究建立在通过未获资助的初步研究建立的坚实基础上，并将侧重于确定一、延伸器部件和功能的特性，二。提供RNA引物和III所需的蛋白质。原产地特定启动所需的蛋白质。重点将放在枯草杆菌复制系统的那些使其不同于大肠杆菌的特征上。这将提供有关细菌复制系统之间在基本成分、机制和调控方面可能发生的变化的重要信息。了解包含两个DNA聚合酶疾病的生物体中DNA复制的生化细节，可能会提供适用于其他双聚合酶复制酶的范例，包括真核生物，在这些真核生物中，POL epsilon和Delta似乎都参与了过程中的染色体复制。

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.