Figure 4

A, B and C. Sequences used in mismatched primer extension and running-start misincorporation assays and examples of analysis. (A) The sequence of the DNA used in each assay type is shown. The underlined nts show the only differences between the two templates. Only one primer was used in the running-start assays and it terminated at the 3′ C nt before the dashes. The four dashes indicate the 4 A nts that must be incorporated before RT incorporates the target nt (denoted by X or Y). (B) Running-start misincorporation of C.T base pair at 2 mM Mg²⁺ or 0.4 mM Zn²⁺. Reactions were performed on the primer-template shown in panel A for the indicated time with a final free concentration of 2 mM Mg²⁺ or 0.4 mM Zn²⁺ (adjusted according to the total concentration of dNTPs in each reaction using the K _d value of Mg²⁺ and ATP). A fixed concentration of dATP = 55 μM was used in all running-start reactions for elongation of the primer to the target site. The concentration of the target nt (dTTP for C.T insertion) in each lane was from l-r: 400, 630, 1380, 2610, and 3660 μM. For other base pair misinsertions noted in the Table 4, the target nt was changed according to the desired misinsertion. (C) Extension of a mismatched primer-template with a C.T 3 terminus, using 2 mM Mg²⁺ and 0.4 mM Zn²⁺. Reactions were performed on the primer-template shown in panel A for the indicated time with the same free cation concentration as above. The concentration of the next correct nt (dCTP) in each lane was from l-r: 50, 100, 200, 400, 630, 1200 and 1870 μM. -E lane corresponds to no enzyme added.