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The electronic form of this document may be cited in the following style:
Human Genome Program, U.S. Department of Energy, DOE Human Genome Program Contractor-Grantee Workshop IV, 1994.

Abstracts scanned from text submitted for November 1994 DOE Human Genome Program Contractor-Grantee Workshop. Inaccuracies have not been corrected.

Studies of T7 DNA Polymerase to Improve the Properties of DNA Sequencing Enzymes

Stanley Tabor, Jeff Himawan, and Charles C. Richardson.
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115

Bacteriophage T7 DNA polymerase modified to remove its 3' to 5' exonuclease activity has properties that are advantageous for DNA sequence analysis. Two of the most important are low discrimination against chain terminating dideoxynucleotides and high processivity in polymerizing DNA. Our studies have been directed towards understanding the mechanism by which T7 DNA polymerase achieves these properties, with the goal of improving it as well as other DNA polymerases for use in DNA sequencing.

T7 DNA polymerase incorporates dideoxynucleotides more efficiently than all known DNA polymerases, not discriminating against them in the presence of manganese. A consequence of this are bands of uniform intensities after gel electrophoresis, a major advantage for DNA sequence analysis. In contrast, two other polymerases used for DNA sequencing, Klenow fragment of E. coli DNA polymerase I and Taq DNA polymerase, discriminate against dideoxynucleotides up to one thousand fold. This results in marked variability in band intensities. This dramatic difference is surprising in light of the fact that all three belong to the same related family of polymerases. We have been using a number of approaches in an attempt to understand the relevant differences that account for this. In one approach we have made use of the fact that T7 DNA polymerase is required for the replication of phage T7 to isolate mutant phage that grow in the presence of dideoxynucleosides. The location of mutations in T7 DNA polymerase responsible for this phenotype reveals the domain where the polymerase interacts with the ribose moiety of the dNTP. In another approach, we are interchanging domains of T7 DNA polymerase with those of Klenow fragment and Taq DNA polymerase, and determining the effect each change has on the ability to incorporate dideoxynucleotides. These mutant polymerases have allowed us to define the region responsible for binding the ribose moiety of dNTPs, and have produced a series of potentially interesting hybrid polymerase molecules with altered specificities for dNTPs.

The high processivity of T7 DNA polymerase is achieved by its interaction with accessory proteins, the predominant one being a tight association with E. coli thioredoxin. We are taking a number of approaches to studying the mechanism by which thioredoxin confers high processivity on T7 DNA polymerase. Our results suggest that thioredoxin acts as a "clamp" which together with T7 DNA polymerase encircles the duplex region of a primer-template. These experiments include the effect of thioredoxin on the footprint of T7 DNA polymerase binding to a primer-template, and the effect of mutant thioredoxins on the polymerase activity of wild-type and mutant T7 DNA polymerases. The goal of these studies is to develop mutant thioredoxins and/or T7 DNA polymerases that result in yet stronger interactions with the DNA, that should improve the properties of the enzyme for DNA sequence analysis. Two other T7 accessory proteins, the T7 helicase/primase and DNA binding protein, also interact with T7 DNA polymerase and dramatically improve its processivity under specific conditions.

Knowledge of the structure of T7 DNA polymerase would be extremely helpful for these studies. Towards this end we, in collaboration with Dr. Thomas Ellenberger (Harvard Medical School) have obtained well ordered crystals of the complex of T7 DNA polymerase and thioredoxin. A complete native data set to 2.8 Å has been determined using the Cornell High Energy Synchrotron Source. We are currently undergoing a screen of heavy atom derivatives in order to solve the structure. In addition to providing the structure of the DNA polymerase widely used for DNA sequencing projects today, this would also represent the first structure obtained of a DNA polymerase with its processivity factor, and would provide important clues in understanding the mechanism by which accessory proteins increase the processivity of DNA polymerases.