In vitro reconstitution and study of Bacillus subtilis DNA replication. (360G-Wellcome-091968_Z_10_Z)

Most of what we know about DNA replication initiation and the organization of the replication fork has emerged from studies of the E. coli system where the replication process has recapitulated in vitro. Little is known about the B. subtilis system because of the lack of a complete in vitro system and the crucial differences in initiation and the organization of the replication fork. Yet the B. subtilis replication is more similar to eukaryotic replication with two polymerases PolC and DnaE act ing on the leading and lagging strand analogous to pol delta and pol alpha, respectively. This proposal aims to recapitulate the oriC-driven B. subtilis complete replication process in vitro. My lab has been working in this area and we are now in possession of all the proteins required as well as unique tools, resources and expertise to achieve this task. Initially we will use it to reveal the molecular mechanism of oriC-replication initiation by the DnaA-DnaD-DnaB proteins and then the molecula r details that underpin the two-polymerase organization of the replication fork. The development of this in vitro system will also be an invaluable tool for future studies of several other crucial differences between B. subtilis and E. coli replication.