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 3

DNA synthesis activity of minimal reconstituted T4 replisomes across a minifork containing a random sequence. The minifork was prepared with the plasmid p-Empty and thus contains a random sequence to be replicated. gp43, gp41, and gp59 are the DNA polymerase, the helicase and the helicase loader of bacteriophage T4, respectively. (A) Gp43 was incubated with the minifork, alone (lane 2), with gp41 (lanes 3–8) or with gp41 and gp59 (lanes 9–15). The reaction was quenched at various times and the samples were loaded on a denaturing sequencing gel. (a) corresponds to the radiolabelled p821 primer. (b) corresponds to radiolabelled p821 extended by 15 nts up to the base of the 5′ ss tail of the lagging strand template. (c) corresponds to the radiolabelled p821 extended up to the end of the leading strand template after strand displacement DNA synthesis. The (a), (b), and (c) DNAs are also shown in the context of the minifork on the right side of the figure. Major transient intermediate products are indicated by backward arrows. The four sequencing reactions (A, T, C, and G) of the leading strand template of the minifork are shown on the left side of the figure. (B) The minifork was incubated with Klenow fragment alone (lanes 2 and 5), together with gp41 (lanes 3 and 6) or with gp41 and gp59 (lanes 4 and 7) for 20 minutes. The reaction products were resolved on a denaturing sequencing gel. Two amounts of Klenow fragment were tested (1 mU/μL, lanes 2–4; 10 mU/μL, lanes 5–7). (a) and (c) are as in 3A.