Regulation of DNA replication is of central importance to the life cycle of a cell. Since bacteria plasmids are small DNA molecules that are maintained at a fixed copy number, they provide useful model systems to study the mechanisms and regulation of DNA replication. The presence of transposons,antibiotic resistance and toxin genes have made studies of plasmid biology of considerable medical significance. We are studying the molecular mechanisms that are involved in the replication of pT181, a small, multicopy plasmid from Staphylococcus aureus. The plasmid pT181 consists of 4437 basepairs, encodes resistance to tetracycline and replicates by a rolling circle mechanism. Replication of pT181 requires the plasmid-encoded replication initiator protein, RepC. The long-term objectives of this project are to understand the DNA-protein interactions involved in the initiation and termination of pT181 DNA replication. A number of experiments will involve the use of an in vitro DNA replication system that is specific for the pT181 plasmid. The regions within the pT181 leading strand origin that are required for the initiation and termination of DNA replication will be identified by deletion analysis as well as by site-directed mutagenesis. The DNA sequences that constitute the pT181 lagging strand origin will be also identified by deletion analysis. In vitro replication experiments will be carried out in the presence of the chain-terminating inhibitor dideoxy TTP to localize the start-site of the lagging strand synthesis. The pT181 initiator protein, RepC, has sequence-specific DNA binding, DNA topoisomerase and DNA replication activities. A number of studies have suggested that different regions of RepC are involved in its DNA binding and DNA relaxation activities. Deletion and site-directed mutagenesis experiments will be carried out using the repC gene and mutant RepC protein will be purified by overexpression in E. coli. The derivatives of RepC will then be tested for their DNA binding, relaxation and replication activities in vitro. Preliminary experiments will also be carried out to isolate the host proteins that are involved in the pT181 leading strand DNA replication. The successful completion of this project should significantly increase our knowledge of the replication of drug resistance plasmids in a Gram- positive pathogen, as well as provide important insights into the mechanisms of DNA replication in general.
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