|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
|15. Structural Analysis of the
T7 DNA Replication System and Further Development of its Use in DNA Sequencing
Stanley Tabor and Charles Richardson
DNA polymerases play an essential role in current methods of DNA sequencing, which require the efficient synthesis of DNA using the four natural nucleotides as well as analogs such as fluorescently-labeled nucleotides and chain-terminating dideoxynucleotides. We have been characterizing the structure and function of DNA polymerases in order to modify those properties that are important for DNA sequencing. Our work has focused on the DNA polymerases of the Pol I family, that includes T7 DNA polymerase and Taq DNA polymerase. We, in collaboration with Sylvie Doublié and Thomas Ellenberger, recently determined the 2.2 crystal structure of T7 DNA polymerase locked in a replicating complex with a dideoxy-terminated primer-template, an incoming dNTP, and the processivity factor thioredoxin1. We are using this structure to design and characterize mutations in the active site of T7 and Taq DNA polymerases that have altered specificity for analogs with modifications in the sugar moiety (e.g. dideoxynucleotides, ribonucleotides and 3' fluoro derivatives) and bases containing bulky fluorescent substituents.
One property that distinguishes T7 DNA polymerase from the thermophilic DNA polymerases used for DNA sequencing and amplification is its high processivity. This is achieved by the binding of its processivity factor, E. coli thioredoxin, to a unique 74 residue domain that acts as a flexible tether to keep the polymerase bound to DNA. While this domain is unique to T7 DNA polymerase, it is modular in that it can be transferred to other homologous polymerases by gene fusion to generate hybrid enzymes that have dramatically increased processivity2. The crystal structure of the T7 DNA polymerase complex suggests that thioredoxin is acting to stabilize the region it binds to, allowing a number of basic residues to interact electrostatically with the DNA backbone to prevent dissociation. We are characterizing mutations in this region in order to further define the critical structural features and to engineer new DNA polymerases that have increased processivity.
The complex of T7 DNA polymerase and T7 helicase/primase synthesize DNA with high efficiency. We have been optimizing reactions carried out by these enzymes in combination with other T7 replication proteins. Conditions have been developed in which DNA synthesis is exponential. Using one pg of plasmid DNA as template, a 15 min reaction can produce 10 g of product DNA, corresponding to a 10 million-fold amplification. DNA synthesis is nonspecific; the entire plasmid is replicated. We are exploring the use of this amplification reaction to produce BAC and plasmid DNA for use in DNA sequencing reactions. This in vitro synthesis of DNA may be an attractive alternative to the current methods that rely on in vivo production in bacterial cells for the automated preparation and purification of DNA templates.
This work is funded in part by DOE grant DE-FG02-96ER62251 (Stanley Tabor, P. I.)
1 Crystal Structure of Bacteriophage T7 DNA Polymerase Complexed to a Primer-Template, a Nucleoside Triphosphate, and its Processivity Factor Thioredoxin. Sylvie Doublié, Stanley Tabor, Alexander Long, Charles C. Richardson and Tom Ellenberger, Nature, 391, 251-258 (1998).
2 The Thioredoxin Binding Domain of Bacteriophage T7 DNA Polymerase Confers Processivity on Escherichia coli DNA Polymerase I. Ella Bedford, Stanley Tabor and Charles C. Richardson, Proc. Natl. Acad. Sci. USA 94, 479-484 (1997).
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