With the exception of plasmid copy control, the mechanisms regulating initiation of DNA replication are still to be defined. The goal of this proposal is to understand how initiation of DNA replication is regulated by cis-acting elements that are located outside of the origin of replication. The present studies will focus on cmp, a cis-acting enhancer of plasmid replication which is located about 1 Kilobase away from the plasmid's origin of replication. The effects of cmp on plasmid replication ( demonstrated by plasmid competition studies and by analysis of exponential replication rates) appear to derive from a change in the efficiency with which the plasmids's replication origin interacts with its initiator protein. cmp activity is orientation-independent and can be detected over a wide range of distances from the origin. cmp is recognized by a site-specific DNA-binding protein 9CBF) encoded by the bacterial host. The goal of this proposal is to understand the molecular mechanism by which cmp enhances plasmid replication and to define the role of host factors in cmp enhancing activity. To achieve this goal, genetic and biochemical studies willbe performed, often in parallel. Deletion analysis of cmp DNA will define the minimal sequence for cmp and the DNA site for CBF binding within cmp will be identified by footprinting. In vivo, cmp activity will be studied using a replication assay; footprinting and nicking assays will define the effect of cmp on the interaction between replication origin and initiator protein. The role of CBF will be studied by inactivating the gene encoding CBF and determining the effect of the mutation on plasmid replication. Purified CBF protein will be obtained to design appropriate probes to screen a library of bacterial chromosomal DNA. Host involvement will be also investigated by isolating host mutants that suppress the plasmid's Cmp defect. The regulatory interaction between cmp and ori may be mediated by a protein-protein bridge involving CBF and the initiator protein, with the intervening DNA looping out. Formation of such a protein complex will be investigated by chromatographic and electrophoretic techniques, and the configuration of the protein-bond plasmid molecules will be examined by electron microscopy. Interactions between cmp and other cis-acting elements in the plasmid and/or other plasmid encoded factors will also be addressed by genetic studies involving isolation of plasmid-linked second-site mutations that suppress the Cmp-phenotype. A knowledge of the loaf-range regulatory interactions in the activity of cmp will contribute to our understanding of how initiation of DNA replication is regulated by cis-acting elements distantly located from the origin of replication. cmp will provide a novel model system to investigate "action at a distance (along DNA)" in general
