The objective of the overall project is to elucidate the biochemical mechanism gDNA replication. This process is complex involving at least 14 proteins and must be studied initially by analyzing the functional activities of the individual proteins. During the past year our goal has been to understand the function of the dnaG and dnaZ gene products, in addition to other proteins, the elongation factors I and III. To probe the unwinding of duplex DNA at the replication fork in prokaryotes, we have made tenylate-primer molecules of DNA whose sequence is known. These molecules form a "replication fork" or stable "Y" structure, and will allow us to test the ability of proteins to promote DNA synthesis into the duplex region of the molecule. The proteins to be tested include the E. coli rep protein and phi X174 cisA protein, both which have been implicated in the unwinding of DNA during phi X174 RFI replication. During the past year we will study the speed, processively, and accuracy of DNA synthesis catalyzed by DNA polymerase III in the presence and absence of these replication proteins. Overall, the next year should allow us to obtain a clearer understanding of the mechanism of DNA replication.