FIG 4.

Substrate specificity of Tk-EshA. (A) Native PAGE demonstration used in the quantitative helicase assay. Tk-EshA was added to assay mixtures containing 2 pmol 5′ overhung (upper panel), 3′ overhung (middle panel), or blunt-end (lower panel) DNA labeled with 5′ IRDye 700. The amount of Tk-EshA was 0, 5, 10, 20, 40, or 60 pmol. *, fluorescent label; S, single-stranded DNA; D, dsDNA of each substrate; T, trapped dsDNA. Because the substrates were trapped by the trap DNA when unwound by Tk-EshA, the position of the substrate band changed. (B) Activities of Tk-EshA for various substrates (forked, 5′ overhung, 3′ overhung, and blunt-end DNAs) relative to that for forked DNA, set to 100%.