Elevated nucleotide turnover by Y831A Pol ε. DNA synthesis and dTTP turnover by Pol ε was measured on poly(dA)/oligo(dT) substrate. Reactions were performed as described in Methods, using 0.13 U of each enzyme for 20 μl reactions. (A) Analysis of polymerase reaction by TLC in 1 M LiCl running buffer. Lanes 1, 2, 3, 4: wild-type Pol ε at 0, 3, 7 and 15 minutes of reaction, respectively. Lanes 5, 6, 7: reactions with Y831A Pol ε at 3, 7 and 15 minutes, respectively. Positions of unincorporated label, label in DNA and dTMP are shown by arrows. (B) Analysis of polymerase reaction by TLC in 0.4 M LiCl running buffer. Lane assignment is the same as in (A). (C) Plot of time-course of DNA synthesis and dTMP turnover by wild-type Pol ε. Open circles connected by solid line represent dTMP retained into DNA; open rectangles connected by a dashed line represent excised dTMP. (D) Plot of time-course of DNA synthesis and dTMP turnover by Y831A Pol ε. Symbols are the same as in (C).