Our goal is to define the molecular mechanisms common to all replication systems. The approach is to characterize the proteins involved in DNA replication using genetic, biochemical, and structural approaches and to identify the functional assemblies that coordinate the reactions. The replication system of bacteriophage T7 is advantageous for this approach since a T7 replisome consists of only a few proteins most of which are encoded by the phage, and yet the replisome functions in a manner similar to those found in more complex systems. Crystal structures of T7 DNA polymerase, T7 helicase-primase, T7 single-stranded DNA (ssDNA) binding protein, and E. coli thioredoxin (processivity factor) provide insight into their functions and suggest future studies. Studies of the contacts of the thioredoxin-binding domain of the thumb of the polymerase with DNA and with residues in the polymerase will elucidate the mechanisms of processivity, DNA partitioning between the polymerase and exonuclease sites, and the role of the thioredoxin binding domain in providing a shared docking site for the replication proteins. The interaction of the gene 2.5 ssDNA binding protein with DNA and with the helicase-primase and polymerase will be examined as will its ability to mediate homologous base pairing. In vitro mutagenesis of the gene 4 helicase will be used to examine oligomerization of the protein, nucleotide binding and hydrolysis, and coupling of nucleotide hydrolysis to unwinding of DNA. The interaction of the Cys4 zinc motif of the gene 4 primase with the catalytic core of the protein to achieve sequence-specific recognition of ssDNA will be studied and the role of the zinc motif in delivering the RNA primer to the polymerase examined. The zinc motif interacts with the RNA polymerase domain of an adjacent subunit within the helicase hexameric assembly. This interaction as well as that of the primase domain with the helicase domain is important in coordinating primase and helicase activities. The four T7 proteins function as a replisome in a mini-circle DNA replication system to mediate coordinated synthesis. Studies using this system will examine the coupling of leading and lagging strand synthesis, the formation of a loop of lagging strand DNA, and the recycling of the lagging strand polymerase.
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