Bacteria do not have mitosis but their genome segregates with fidelity during cell division. This is thought to take place by the attachment of DNA to the membrane. In vitro, the replicative origin of the Escherichia coli chromosome (oriC) selectively attaches to membranes when it is hemimethylated at Dam methylase recognition sites, which suggests that segregation is determined by events that are closely tied to initiation of replication. This proposal is to study the nature of this attachment and its cyclical behavior, and to extend the study to plasmids and other bacterial species. The experiments fall into five related areas: 1. Determination of the Dam recognition sites that must be hemimethylated for oriC to bind to membranes. 2. Isolation and characterization of """"""""membrane receptor"""""""" proteins. 3. In vivo protein interactions with oriC DNA, studied by treatment of intact cells with dimethyl sulfate. 4. Plasmid Segregation. Do centromere-like sites or other regions of low copy plasmids (F, P1) bind specifically to membranes? Do they encode their own """"""""membrane receptors""""""""? 5. Does hemimethylation play a role in origin-membrane binding in bacteria that do not have Dam methylase?
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