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 1
Strategy of the preparation of model replication miniforks. (a) The left and right sides of the figure correspond to the strategy used to build the ss leading and lagging strand templates of the minifork, respectively. The ss leading and lagging strand templates are combined with the radiolabelled p821 primer to assemble the minifork by strand hybridization. Replication miniforks are prepared in three consecutive steps. The first step (Step 1: PCR) consists of a PCR using plasmids containing a random or a TNR sequence (shown as a black rectangle) and oligonucleotides that flank the random sequence or the TNR unit. For each PCR, one of the oligonucleotides (p1033/4ps (colored in blue) for the preparation of the ss leading strand template, and (50T/4ps)/p867 (colored in red) for the preparation of the ss lagging strand template) carries 4 phosphorothioate linkages (represented as filled blue and red spheres for p1033/4ps and (50T/4ps)/p867, resp.) at its 5′ end. After PCR, the ds PCR products are digested by the T7 exonuclease that specifically degrades the DNA strand (colored in green or orange for the preparation of the ss leading or lagging strand template, resp.) that does not contain the phosphorothioate linkages (Step 2: T7 exonuclease digestion). The minifork (shown in a rounded rectangle) is assembled by hybridization of the ss leading and lagging strand templates and the radiolabelled p821 primer (in green) (Step 3: Hybridization). A gap of 15 nts exists between the 3′ end of the p821 primer and the base of the ss tail of the lagging strand template to facilitate the assembly of the DNA polymerase at the p-t junction. (b) A minifork containing n repeats of 5′CTG is shown. The repeats are located on the leading strand template.
