Helix–hairpin–helix motifs confer salt resistance and processivity on chimeric DNA polymerases

Abstract

Helix-hairpin-helix (HhH) is a widespread motif involved in sequence-nonspecific DNA binding. The majority of HhH motifs function as DNA-binding modules with typical occurrence of one HhH motif or one or two (HhH)2 domains in proteins. We recently identified 24 HhH motifs in DNA topoisomerase V (Topo V). Although these motifs are dispensable for the topoisomerase activity of Topo V, their removal narrows the salt concentration range for topoisomerase activity tenfold. Here, we demonstrate the utility of Topo V's HhH motifs for modulating DNA-binding properties of the Stoffel fragment of TaqDNA polymerase and Pfu DNA polymerase. Different HhH cassettes fused with either NH2 terminus or COOH terminus of DNA polymerases broaden the salt concentration range of the polymerase activity significantly (up to 0.5 M NaCl or 1.8 M potassium glutamate). We found that anions play a major role in the inhibition of DNA polymerase activity. The resistance of initial extension rates and the processivity of chimeric polymerases to salts depend on the structure of added HhH motifs. Regardless of the type of the construct, the thermal stability of chimeric Taq polymerases increases under the optimal ionic conditions, as compared with that of TaqDNA polymerase or its Stoffel fragment. Our approach to raise the salt tolerance, processivity, and thermostability of Taq and Pfu DNA polymerases may be applied to all pol1-and polB-type polymerases, as well as to other DNA processing enzymes. Many gene regulatory proteins contain small, discrete structural motifs that use either ␣-helices or ␤-strands for specific DNA binding. Among these are the helix-turn-helix, zinc finger, leucine zipper, and helix-loop-helix motifs (1). The motifs may arise in different molecular contexts, which is believed to occur because of either divergent evolution by means of gene duplication and insertion or structural convergence by means of the effects of selective pressures on protein function. Other proteins bind DNA by using sequence-nonspecific interactions. They belong to various protein families, such as nucleases, N-glycosylases, ligases, polymerases, and helicases that are essential for the protein-mediated synthesis and DNA repair. This compact structure with a well defined hydrophobic core mirrors the symmetry of the DNA-double helix and facilitates strong DNA binding. Protein-DNA contacts do not involve DNA bases but rather a sugar-phosphate chain, making proteins with HhH motifs able to bind DNA in the nonsequence-specific manner.A unique number of HhH motifs has been identified in DNA topoisomerase V (TopoV; refs. 6-8). The 684 C-terminal amino acids (from a total of 984 amino acids) are organized into 12 repeats of Ϸ50 amino acids long each (9). All repeats consist of two similar HhH motifs. We demonstrated earlier that Topo V proteins lacking different parts of an HhH superdomain remain fully active in relation to supercoiled DNA but become sensitive to salts (9, 10). Thus, HhH motifs play a crucial role in Topo V interactions with DNA, which a...

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