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Helix-hairpin-helix motifs to manipulate properties of dna processing enzymes

United States Patent Application

View the Complete Application at the US Patent & Trademark Office
The utility of Topo V's HhH motifs for modulating DNA binding properties of a DNA processing enzyme, such as Taq DNA polymerase or PfuDNA polymerase, is demonstrated. In one embodiment, an amino acid residue comprising one or more HhH domains derived from Topoisomerase V, linked to either the NH.sub.2-terminus or COOH-terminus of a Taq polymerase fragment significantly broadens the processivity and/or salt concentration range of polymerase activity. The specific activities of the chimeric polymerases are not affected by added HhH motifs. Depending on the type of the construct, the thermal stability of chimeric polymerases increases or remains the same as that of Taq DNA polymerase or its Stoffel fragment. This invention further provides a method of increasing the salt tolerance of Taq polymerases. The methods of this invention may be applied to all pol A-type DNA polymerases, as well as to other DNA processing enzymes.
Slesarev, Alexei (Boyds, MD), Pavlo, Andrey (Gaithersburg, MD), Kozyavkin, Sergei (Germantown, MD), Pavlova, Nadezhda (Gaithersburg, MD)
10/ 514,342
May 14, 2003
[0001] This work was supported in part by U.S. Department of Energy and National Institutes of Health grants (DE-FG02-98ER82577, 00ER83009, R44M55485, R43HG02186) to S. Kozyavkin and A. Slesarev.