The composition, structure, and function of multiprotein complexes that catalyze different steps of chromosomal DNA replication in Salmonella typhimurium will be examined through analysis of extragenic suppressor mutations that reverse the phenotype of particular replication mutants. Many such suppressors will alter other proteins in the complex in which the original mutant protein acts. Therefore, these other proteins will be identified genetically and inferences about the physical and functional relationships among the proteins in these complexes can be made. Salmonella has been found to possess replication genes functionally equivalent to each of the familiar replication genes of E. coli. Suppressors will be sought for mutations in genes whose products are associated with the polymerase III elongation complex (dnaE, N, X, Z) or, in a parallel set of experiments, genes involved with the so-called primosome, thought to be responsible for the priming of Okazaki fragments (dnaB, C, G, ssb). Suppressors will be identified within Salmonella genes cloned in phage lambda by a novel """"""""red plaque"""""""" assay used in conjunction with lambda-Salmonella genomic libraries prepared from specific mutants. For example, a library prepared from a dnaG mutant will be mutagenized and screened for red plaque phage on a dnaG host, in order to find suppressors of dnaG. Suppressor phase will be subjected to further genetic and physical analysis to establish the identity of the gene mutating to the suppressor form, and to initial genetic and biochemical analysis aimed at understanding the basis of suppression. In the long term these experiments will contribute to a detailed understanding of DNA replication at the molecular level. In addition, because these experiments entail the molecular cloning of each replication gene, the experiments may lead to investigations of the regulation of replication genes and possibly even the regulation of replication itself. Finally, these studies provide a practical model for the investigation of other complex, multiprotein processes in bacteria.
