Fig. 4.

(A) Misincorporation during DNA replication by mutants at residue Asp¹²¹ of ϕ29 DNAP. The assay was performed as described in Materials and Methods, using as substrate the 5′-labeled hybrid sp1/sp1c+6 (depicted on top of the figure) and the indicated concentration of dATP. After incubation for 5 min at 25 °C, samples were analyzed by 7 M urea/20% polyacrylamide gel electrophoresis and autoradiography. The position corresponding to the unextended primer (15mer) and to either extended (17mer, 19mer, and 21 mer) or degraded (4mer) products is indicated. (B) Polymerization/exonuclease-coupled assay. The assay was performed as described in Materials and Methods using the 5′-labeled hybrid sp1/sp1c+6 as substrate and the indicated concentration of the four dNTPs. After incubation for 5 min at 25 °C, samples were analyzed by 7 M urea/20% polyacrylamide gel electrophoresis and autoradiography. Polymerization or 3′-5′ exonucleolysis is detected as an increase or decrease, respectively, in the size (15mer) of the 5′-labeled primer. (C) Translocation assay. The assay was performed as described in Materials and Methods using the 5′-labeled hybrid sp1/sp1c+13A (15mer/28 mer). After incubation for 5 min at 25 °C, samples were analyzed by 7 M urea/20% polyacrylamide gel electrophoresis and autoradiography. The position corresponding to the primer (0) and +1 and +2 extension products is indicated.