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 6
At low concentrations of dNTPs, 5′CTG repeats create a greater impediment than 5′CAG repeats to the gp41-gp43-gp59 replication trio. (a) Miniforks containing 17 5′CTG repeats (left panel) and 23 5′CAG repeats (right panel) on the leading strand template were incubated with gp43, gp41, and gp59 for 10 min at various concentrations of dNTPs, and the reaction products were resolved on a denaturing sequencing gel. Each of the four dNTPs is present at the following concentration (in μM): 0 (lanes 1 and 14); 0.71 (lanes 2 and 8); 1.42 (lanes 3 and 9); 2.84 (lanes 4 and 10); 5.68 (lanes 5 and 11); 11.36 (lanes 6 and 12); 22.72 (lanes 7 and 13). Three of the four sequencing reactions (A, C, G for the minifork containing the 5′CTG repeats, left; T, C, G for the minifork containing the 5′CAG repeats, right) of the leading strand template of the minifork are shown on the right side of the dNTP titrations. The TNR unit is designated by a double-headed arrow. The lines along which DNA synthesis profiles at 2.84 μM dNTPs (lanes 4 and 10) have been established are shown on the side of each panel and are divided into two parts labelled “prior to TNR” (from position 0 to 165 for the miniforks containing the 5′CTG repeats or position 0 to 170 for the miniforks containing the 5′CAG repeats) and “across and past TNR” (from position 160 to 250 for the miniforks containing the 5′CTG repeats or position 170 to 260 for the miniforks containing the 5′CAG repeats). (b) DNA synthesis profile at 2.84 μM dNTPs prior to the TNR unit from position 0 to 165 for the miniforks containing the 5′CTG repeat (left panel) and from position 0 to 170 for the miniforks containing the 5′CAG repeats (right panel). (c) DNA synthesis profile at 2.84 μM dNTPs across and beyond the TNR unit from position 160 to 250 for the miniforks containing the 5′CTG repeat (left panel) and from position 170 to 260 for the miniforks containing the 5′CAG repeats (right panel). a.u.: arbitrary unit.
