Are Membrane Proteins Directly Involved in Prokaryotic DNA Replication?

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The proposed investigations will critically analyze the binding characteristics of several membrane proteins that complex with single or ouble stranded cloned DNA inserts, derived from the B. subtilis origin of replication. One 65kD protein that binds strongly to two adjacent origin inserts, but not to flanking origin regions or non-origin DNA inserts will be examined in detail. The protein will be purified, its membrane form confirmed, and a determination will be made of specific sequences involved in the protein-DNA binding. The relationship of this protein to various dna initiation genes will be assessed by in vitro complementation analysis using DNA/membrane subcomplexes extracted from mutants conditionally defective in such dna genes. The dnaB gene will be of particular interest to study because it has already been shown to encode a membrane protein with affinity for replication origin DNA sites. Functional analyses will be done with antibodies against the 65kD protein, to measure their ability to block replication. Although the function of membrane associated DNA binding proteins in the process of replication is presently uncertain, their importance in the process is established. The results of the novel and rigorous experiments described in this proposal are certain to provide important new information on the sequence of events that occur during chromosome replication.