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 4

At high concentrations of dNTPs, 5′CAG repeats create a greater impediment than 5′CTG repeats to the gp41-gp43-gp59 replication trio. (A) The leading strand template of the minifork carries 17 repeats of 5′CTG. Three of the four sequencing reactions (A, C, G) of the leading strand template of the minifork are shown on the right side of the figure. The TNR unit is designated by a double-headed arrow. The minifork was incubated with gp43 alone (lane 2), with gp43 and gp41 (lanes 3 and 4), or with gp43, gp41 and gp59 (lanes 5–7). The reaction was quenched at various times and the samples were loaded onto a denaturing sequencing gel. (a), (b), and (c) are as in Figure 3. The primers extended by 14 to 16 nts are shown in the bracket labelled “prior to TNR” and are used to estimate the rate of the gp41-gp43-gp59 replication trio before the TNR unit. The elongation products that contain the full-length TNR are shown in the bracket labelled “past TNR” and are used to estimate the rate of the minimal reconstituted T4 replisome across the TNR unit. (B) The graph shows the decrease of intensity of primers elongated by 14 to 16 nts (indicated by the bracket labelled “prior to TNR” in 4) as a function of time for the miniforks containing 5′CTG or 5′CAG repeats on their leading strand template. (C) The graph shows the accumulation of DNA synthesis products that contain the full-length TNR unit (indicated as a bracket labelled “past TNR” in A) as a function of time for the miniforks containing 5′CTG or 5′CAG repeats on their leading strand template. (D) Black bars: ratio between the amount of primers extended by 14 to 16 nts measured with miniforks containing 5′CTG repeats and the amount of primers extended by 14 to 16 nts measured with the miniforks containing 5′CAG repeats at different times. White bars: ratio between the amount of DNA synthesis products that contain the full-length CAG unit and the amount of DNA synthesis products that contain the full-length CTG unit at different times. The error bars correspond to the standard deviation calculated from at least two independent experiments. B and C were obtained from quantifying the experiment presented in 4(A), and the experiment was performed in parallel with a minifork containing 23 5′CAG repeats. a.u.: arbitrary unit.