Our goal is to define mechanisms common to all replication systems by studying the process in the relatively simple replication system of bacteriophage T7. We will characterize the protein assemblies by genetic, biochemical, and structural methods. Movement of the T7 DNA replication fork involves only four proteins, three of which are encoded by the phage. This simplicity has made possible (i) the reconstitution of a replisome in which DNA synthesis is coordinated and nascent Okazaki fragments are found in replication loops, (ii) the determination of the crystal structures of all four proteins, and (iii) the visualization of replisomes by single- molecule techniques. T7 gene 5 DNA polymerase contains a unique segment (TBD within the thumb to which E. coli thioredoxin binds to increase processivity. The interaction of thioredoxin with the TBD, in turn, creates two basic loops to which the gene 4 helicase and the gene 2.5 ssDNA binding protein dock. Both these proteins have an acidic C-terminal tail bearing a C-terminal phenylalanine. Studies are designed to characterize this three-way switch. The essential phenylalanine has allowed the identification of contact points within the gene 5 protein. Altered gene 4 helicases provide an approach to understanding the sequential hydrolysis of dTTP, translocation on DNA, and the basis for dTTP specificity. Single molecule techniques allow us to visualize the movement of hexameric helicases on ssDNA. We are examining the interaction of the zinc motif of the primase with recognition sites in DNA. Single molecule experiments reveal pausing of the replisome during primer synthesis. We speculate that a trans mode of primer synthesis involving adjacent subunits plays a role in the process. During DNA synthesis the interaction of helicase with polymerase involves contacts other than the C- terminal tail, an interaction we hope to elucidate. Replicating polymerases can exchange with polymerases in solution without loss of processivity. We are exploring the mechanism by which this reaction occurs. Complexes of the T7 proteins with DNA are being crystallized. Gene 1.7 protein enables dideoxynucleosides to stop T7 growth and E. coli uridine/kinase can inhibit T7 growth. Both observations are under study. The rationale for studying the replication system of bacteriophage T7 is based on the premise that the molecular events found in this relatively simple system can be extended to more complex organisms. Such information provides a rational basis for curtailing abnormal replication and recombination in neoplastic cells. The identification of inhibitors of the proteins involved can provide novel chemo-therapeutic agents. In addition, the proteins of DNA replication can, themselves, often be used as reagents to dissect other processes. ? ? ?
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