This is an ambitious program of x-ray crystal structure analyses, aimed at understanding the mode of action of the E. coli T7 DNA polymerase. T7 is important, in that it is one of the simplest of all replicative polymerases. It is a heterodimer consisting of an 80 kD gene 5 protein and a 12 kD E. coli thioredoxin. Both polymerase activity and the 3'-5' exonuclease activity of the holoenzyme reside in the gene 5 subunit. Thioredoxin is a processivity factor stabilizing interaction of the polymerase with DNA, increasing processivity from 10-50 bases to several thousand bases before the polymerase falls off the DNA template. In contrast to T7, most other replicative polymerases are large multiprotein complexes. Although T7 catalyzes DNA synthesis from primed single-stranded DNA in vitro, replication in E. coli requires assistance of several other phage-endcoded proteins. Among them, gene 2.5 protein, a dimer of 25.5 kD subunits, binds ssDNA with high affinity and greatly accelerates annealing of homologous DNA strands. It assists lagging-strand synthesis and contributes to the coupling of leading and lagging-strand syntheses. Specific projects planned include crystal structure analyses of: (1) ternary complexes of T7 polymerase, primer/template DNA, and mononucleotide, employing each of the four mononucleotides in turn: dGTP, dATP, dCTP and dTTP, in each case with the proper DNA template; (2) the """"""""apopolymerase"""""""" alone, without bound DNA, (3) a complex of polymerase and primer/template having the wrong mononucleotide incorporated at the 3' position, or a 3' mismatch (following a search for the most stable mismatched complexes) and (4) the complex of T7 polymerase or one of its domains with the T7 gene 2.5 protein. This entire project represents a long-term collaboration with Prof. Charles Richardson, also of Harvard.
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