Mechanism of Termination of DNA Replication

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DESCRIPTION (provided by applicant): This application describes experiments designed to address two significant questions regarding the mechanism of replication fork arrest in prokaryotes and an extensive set of experiments to identify and study the mechanism of action of proteins involved in replication termination in yeast rDNA and thus represents a transition of work from prokaryotes to eukaryotes. First, the mechanism of conditional, ppGpp-dependent fork arrest at the replication checkpoints of Bacillus subtilis will be analyzed to investigate if long range DNA-DNA interaction promoted by the replication termination protein (RTP) is the mechanism that converts a weak replication pause site to an efficient terminus. The preceding experiments will make use of fluorescent in situ hybridization, formaldehyde crosslinking and DNA microarrays as the principal experimental tools. Second, the mechanistic aspects of interaction between Tus and DnaB of Escherichia coli will be investigated by reverse 2-hybrid analysis to identify mutations in the helicase DnaB that allow the helicase to pass through the barrier of the replication terminus. These mutants and their suppressors in Tus will be used, along with wild type proteins to study Tus-DnaB interaction by EM and 3D image reconstruction. The main question that the principal investigator is trying to address is how does the contact between Tus and DnaB block the helicase activity of the latter and whether the physical contact inhibits the DNA-dependent ATPase activity of DnaB. A novel replication activation-based 2-hybrid system will be used to identify if the Ter-Tus complex or the dif-XerC/D complex interact with proteins located at or near the z ring to localize and hold the Ter at the cell center during chromosome replication and segregation. Finally, a multifaceted approach involving yeast genetics and molecular biology will be used to identify the proteins that interact with Fob 1 to arrest forks at yeast rDNA. The principal investigator will also try to identify the component of the yeast replication apparatus that interacts with the termination complex.