DNA replication is a key genetic process in all living organisms. In Salmonella typhimurium, this process is catalyzed through the cooperation of more than 20 proteins. The long term objective is to define how these proteins cooperate, with particular reference to direct physical interactions in complexes. The goals are to identify which pairs of proteins interact, and at a more detailed level to identify the individual determinants within these proteins that are responsible for the interaction. By examining DNA replication proteins for interactions in pairwise fashion, an overall picture of the replication apparatus can be developed. In the next period, emphasis will be placed on the following specific aims: 1. To understand how suppressor mutations allow a functional DNA polymerase III to be made in cells lacking one of the normal core subunits of this enzyme, the Epsilon subunit encoded by the dnaQ gene. 2. To identify and study the gene for the Theta subunit of this same enzyme. 3. To characterize the interaction of wild type and suppressor dnaB protein with dnaC protein to determine if the suppressor mutation alters the affinity of the interaction; and if so, to study new mutations of dnaB produced at the same site. 4. To explore the potential of monoclonal antibodies as """"""""surrogate"""""""" protein ligands for the isolation of mutations specifically affected in ligand binding. 5. To study regulatory effects of dnaA mutations on expression of previously derived suppressor alleles in the adjacent gene, dnaN. Different aspects of these studies utilize a variety of techniques, including, among others: extragenic suppression analysis to identify potentially interacting proteins; protein purification and characterization to assess mechanisms of suppression; molecular cloning of suppressor genes in phage Lambda to aid in characterization and identification of the gene product; antibody binding to protein antigens in Lambda plaques to identify antibody-resistant mutants; and gene fusions to aid in dissecting regulatory circuits.
