The role of mismatch repair proteins in origin sequestration

We have shown that DNA replication is tightly coupled to cell growth in wild type E. coli cells. Initiation of replication occurs precisely at the same point in the cell cycle, generation after generation. The molecular mechanisms behind the precise timin g of initiation of replication are not yet understood. We have characterized gene products that are important for maintenance of correct initiation frequency. The DnaA protein is the main actor in the initiation process. It recognizes the origin, separate s the strands of the double helix and recruits the replication machinery. At the time of initiation there is a danger of immediate reinitiation. Reinitiation at new origins is prevented by two different control systems. First, a process called sequestrati on makes newly formed origins unavailable to the initiation apparatus. The sequestration process involves membrane components, SeqA protein and DNA adenine methylation at GATC sites. Second, the DnaA protein is inactivated by a process termed regulatory i nactivation of DnaA (RIDA). The RIDA mechanism is dependent on the Hda protein and the beta clamp of an actively replicating polymerase. The SeqA protein can distinguish newly replicated DNA from old DNA because the new DNA is not yet methylated at GATC sites on the new strand. The mismatch repair protein system also needs to distinguish new from old DNA, and this system also relies on hemimethylation of GATC sites. In this project we investigate the role of mismatch repair proteins in origin sequestrati on. We also investigate the RIDA mechanism through characterization of the mechanism of action of the Hda protein and the beta clamp.