The investigator is interested in understanding how bacterial plasmids are partitioned after cell division. He is using the plasmid pSC101 as a model system for studying the partition process. Previously, he has characterized a par locus on pSC101 that is essential for correct partitioning, and has identified some chromosomally encoded proteins (e.g., TopA, IHF) that can mitigate the effects of deleting par. He has evidence to suggest that the par locus does not encode a protein but rather acts in cis to alter plasmid DNA topology. Dr. Cohen's model is that RepA monomers bind to the origin region, together with DnaA and possibly other host proteins, to form a complex that is involved both in replication and partition (the P/R complex). It is this complex, not the par locus, that interacts with the cytoplasmic membrane during replication. The function of the par locus is to ensure the correct degree of supercoiling that promotes formation of this complex. The long term goal of the proposed work is to determine how RepA participates in replication and partitioning, and to determine how the partition/replication complex interacts with host proteins to mediate partitioning. Specifically, he proposes to (i) use ultracentrifugation , and ultimately X-ray crystallographic analysis of wild type and mutant RepAs (that compensate for lack of a par locus) to determine the relationship between RepA protein structure and its effects on partitioning and replication, (ii) identify and analyze host proteins that affect pSC101 partitioning at a step subsequent to formation of the partition-replication complex, (iii) use the two-hybrid system to identify host genes encoding proteins that interact with the RepA/DnaA/plasmid origin partition-replication complex, and (iv) investigate the temporal and physical relationship between partitioning of post-replicative plasmids, septation and cell division.
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