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J. Biol. Chem., Vol. 279, Issue 18, 18288-18295, April 30, 2004

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DNA-Thumb Interactions and Processivity of T7 DNA Polymerase in Comparison to Yeast Polymerase ^*

Vincent J. Cannistraro and John-Stephen Taylor

From the Department of Chemistry, Washington University, St. Louis, Missouri 63130

The replicative polymerase of bacteriophage T7 is structurally and mechanistically well characterized. The crystal structure of T7 DNA polymerase or gene 5 protein complexed to its processivity factor, Escherichia coli thioredoxin, a primer-template, and a dideoxynucleotide reveals how this enzyme interacts with the 3'-end of the primer-template, but does not show how thioredoxin confers processivity to the polymerase. In the crystal structure highly conserved amino acids Asn³³⁵ and Ser³³⁸ of the thumb subdomain of T7 DNA polymerase are seen to interact with phosphates 7 and 8 of the DNA template strand. Results with a mutant T7 DNA polymerase in which aliphatic residues are substituted for these amino acids and experiments with different length and methylphosphonate-modified primer-templates demonstrate that these interactions are essential for processive synthesis and d(A·T)_n tract bypass. Our data with methylphosphonate-modified DNA suggests that thioredoxin confers processivity to T7 DNA polymerase in part by causing an interaction with the phosphate backbone or minor groove of DNA. Residues Asn³³⁵ and Ser³³⁸ may also function with a nearby helix-loop-helix motif located at residues 339–372 to enclose the DNA during processive synthesis. Our results suggest that this structure must be held close to the DNA by ionic interactions to function. These interactions also allow for DNA sliding but physically block the passage of a 3T bulge in the template. In contrast, yeast polymerase , a polymerase that non-mutagenically repairs cis-syn thymidine dimers, allows the same bulge to slide past its thumb subdomain during synthesis. A relaxed thumb interaction with the DNA could account for the notably low processivity of polymerase .

Received for publication, January 12, 2004

^* This work was supported by National Institutes of Health Grant CA-40463. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Chemistry, Washington University, One Brookings Dr., Campus Box 1134, St. Louis, MO 63130. Tel.: 314-935-6721; Fax: 314-935-4481; E-mail: taylor{at}wustl.edu.

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