Helix-hairpin-helix motifs confer salt resistance and processivity on chimeric DNA polymerases.
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 NH(2) 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.
D020816:Amino Acid Motifs; D001483:Base Sequence; D001622:Betaine; D001665:Binding Sites; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D005971:Glutamates; D066298:In Vitro Techniques; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride; D013379:Substrate Specificity; D019914:Taq Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D005971:Glutamates; D011993:Recombinant Fusion Proteins; D001622:Betaine; D012965:Sodium Chloride; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase; C082698:DNA topoisomerase V; D004264:DNA Topoisomerases, Type I