We intend to characterize the numerous enzymes and proteins which we have identified and isolated as participants in the initiation of a cycle of replication of the E. coli chromosome at its origin (oriC) and the initiation of nascent chains at replications forks. These are some of our specific objectives: (i) Described in molecular detail how the origin of the E. coli chromosome is recognized by dnaA protein and so configured to create a bubble for the priming of replication. (ii) Discover how the dnaB helicase, with the aid of dnaC protein, is positioned to create bidirectional forks of replication. (iii) Determine the mechanisms and varieties of transcription activation of initiation at oriC. (iv) Examine the fate and cycling of the proteins that participate in initiation, the influence of membranes, and of ATP and energy charge. (v) Explore existence of factors (positive or negative) which may serve as signals from the increase in cell mass that commit the cell to initiate a new cycle of replication. (vi) Determine the genetic loci and prepare mutants of the primosomal proteins (n, n', and n"""""""") to evaluate their physiological importance and their operation at the replication forks of the E. coli chromosome. These biochemical studies are essential for revealing the facts and patterns of replication basic to understanding cell growth and cell division in eukaryotic as well as prokaryotic organisms. With a grasp of the biochemical nature of the switch that operates the initiation of replication, will come opportunities to discover the factors that regulate orderly growth and those responsible for uncontrolled proliferation (e.g. cancer) or its premature cessation.
