Dr. Khan is studying the replication of plasmids from Gram-positive bacteria, using pT181 as a model system. The long-term goals of this project are to understand the DNA-protein and protein-protein interactions that are important in pT181 replication and its regulation. It is now well-established that most small multicopy plasmids of Gram-positive bacteria, including pT181, replicate by a rolling circle mechanism. The replication initiator protein of pT181, RepC, has DNA binding an origin-specific, nicking-closing activities. The origin of leading strand replication contains signals for both the initiation and termination of replication. Specific goals include: (i) The nucleotide sequence requirements of the origin, and the role of a particular inverted repeat IRII, will be studied both in vivo and in vitro by site-directed mutagenesis. (ii) A single-strand origin, ssoA, is involved in lagging strand synthesis, and the roles of the conserved folded structure and sequences of ssoA in lagging strand replication will be studied. (iii) The initiators encoded by the plasmids of the pT181 family function as dimers. The role of individual monomers of RepC in its biological activities will be investigated. Heterodimers between two RepC mutants, one lacking the DNA binding and the other the topoisomerase activity, will be generated and tested for reconstitution of the replication activity. The RepC-RepC* complex, an inactivated heterodimer generated after one round of replication in vivo, will be isolated and tested for its various biochemical activities in vitro. A model for the roles of the two RepC monomers in replication and its regulation will be developed. (iv) Finally, crystallization studies of RepC are proposed.
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