FIG. 1.

(A) Overlay of the electrostatic potential and chemical structure of thymine (left) and the nonpolar, isosteric nucleobase analog, 2,4-difluorotoluene (right). (B) Running-start and standing-start DNA substrates. The 28-mer templates have either a T or an F at position 24, shown as a bold N. (C) Running-start and standing-start assays with Taq and human Polκ. Lanes 1 to 3 are running-start experiments, and lanes 4 to 6 are standing-start experiments. Lanes 1 and 4 contain no protein, lanes 2 and 5 contain a template T residue, and lanes 3 and 6 contain a template F residue. The stalling of synthesis by Polκ 1 to 2 nucleotides prior to the end of the DNA template is a feature that is characteristic of this polymerase (10).