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.
