Crystal Structure of a Ternary Complex of T7 DNA Polymerase with DNA and Nucleotide Substrates. Sylvie Doublié, Alexander Long, Stanley Tabor, Charles Richardson, and Tom Ellenberger, Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston MA 02115

We have crystallized a ternary complex of the bacteriophage T7 DNA polymerase with DNA primer-template and nucleotide triphosphate substrates bound in the polymerase active site. The crystal structure of the 108 kDa complex was determined at a resolution of 2.5Å using multiwavelength anomalous dispersion of the selenomethionyl-protein.

The T7 DNA polymerase consists of a 1:1 complex of a phage-encoded gene 5 catalytic subunit and E. coli thioredoxin, which confers high processivity to the polymerase, thereby ensuring efficient replication of the phage genome. The high fidelity and processivity of the T7 holoenzyme rival those of other, more complicated replicative DNA polymerases.

The overall protein fold of the T7 DNA polymerase is similar to those of other Pol I-type polymerases, including the E. coli Pol I Klenow fragment, the Thermus aquaticus DNA polymerase, and a Bacillus stearothermophilus DNA polymerase. However, the T7 enzyme has several regions of unique topology including a thioredoxin-binding domain located at the tip of the thumb, which is required for processive DNA synthesis.

The experimental electron density clearly shows the orientation of the primer-template DNA and the incoming nucleotide triphosphate with respect to active site residues that are conserved in the Pol I family. The structure of the T7 enzyme provides the first glimpse of a replicative polymerase locked in polymerization mode and it will serve as the basis for mechanistic studies addressing the processivity, substrate range, and fidelity of DNA synthesis.

This work is supported by the Lucille P. Markey charitable trust, the National Institutes of Health, and the Department of Energy.