Regulation and organization of chromosome replication and segregation in Escherichia coli.

In Escherichia coli, reinitiation at new origins is prevented by two different control systems. First, a process called sequestration makes newly formed origins unavailable to the initiation apparatus. The sequestration process involves membrane component s, SeqA protein and methylation at GATC sites by Dam methylase. In the second control circuit, the initiator protein, DnaA, is inactivated by the Hda protein and the sliding clamp of the polymerase. The inactivation of DnaA also contributes to regulation of nucleotide synthesis. Binding of SeqA to GATC sites is also important for organization and segregation of daughter chromosomes. We have found that pairs of replication forks are co-localized in the cell and that SeqA forms a left-handed helical structu re that may contribute to sister chromosome cohesion. In this project we will characterize a quorum sensing like regulatory signal that is capable of changing the initiation frequency. The factor will first be purified and identified and then its mechanis m of action characterized. We will also investigate how the RIDA (regulatory inactivation of DnaA) mechanism works through characterization of the mechanism of action of the Hda protein and the beta clamp.