5′CAG and 5′CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs
Figure 7
DNA synthesis profiles and efficiencies at various concentrations of dNTPs. (a) The minifork used here carries a random sequence. The four sequencing reactions (A, T, C, and G) of the leading strand template of the minifork are shown on the right side of the gel. The minifork was incubated with gp43, gp41 and gp59 for 10 min with the indicated dNTP mixture and the samples were loaded onto a denaturing sequencing gel. “4 low” (lane 1) means that the 4 dNTPs are at a low concentration (1 μM). “4 high” (lane 6) means that the 4 dNTPs are at a high concentration (80 μM). The single dNTP at low concentration (1 μM) is indicated on the top of the figure (dATP, TTP, dCTP, and dGTP in lanes 2, 3, 4, and 5, resp.). The three other dNTPs are at 80 μM. The backward arrow points to the TNR insertion site. (b) The gel shown in 7(a) has been quantified and the ratio R is shown for each reaction condition (low [dATP], low [TTP], low [dCTP], and low [dGTP]). R is the ratio between the amount of DNA synthesized past the TNR insertion site under a given condition of [dNTPs] (low [dATP] (lane 2 of Figure 7(a)), low [TTP] (lane 3 of Figure 7(a)), low [dCTP] (lane 4 of Figure 7(a)), or low [dGTP] (lane 5 of Figure 7(a)) and the amount of DNA synthesized past the TNR insertion site at low [dGTP] (lane 5 of Figure 7(a)). The number of each dNTP that has to be incorporated up to the insertion site is also indicated underneath the panel of histograms. The same quantification as that described in 7(b) was performed with miniforks carrying a 5′GTT (c), 5′CAG (d) and 5′CTG (e) repeat. For the miniforks containing a TNR unit, R is the ratio between the amount of DNA synthesized past the TNR unit under a given condition of [dNTPs] (low [dATP], low [TTP], low [dCTP], or low [dGTP]) and the amount of DNA synthesized past the TNR unit at low [dGTP]. The number of each dNTP that has to be incorporated up to the end of the TNR unit is indicated underneath the panel of histograms.
