Figure 4. Head-to-head CMGs unwind dsDNA.

(A) Scheme of the reaction with the double tailed substrate, and two possible processes that could result in unwinding the DNA. The color scheme is the same as in Figure 1. CMG is mixed with the substrate on ice and then incubated at 30° C in the absence of ATP (top) to allow the reaction to reach temperature. 1’ later, ATP is added to allow CMGs to load onto the duplex in opposite orientations and block each other’s progress (middle). At the same time as ATP, Mcm10 is added to initiate the duplex unwinding reaction. 45’ later, an ssDNA trap oligo (orange) is added that quenches further CMG loading (Figure 4—figure supplement 1) and also anneals to the unwound radiolabeled product, creating a forked structure (bottom) that migrates at a distinct position in the gel from the substrate. Unwinding may occur either by: strand shearing (left arrow), or by strand expulsion (right arrow) to form CMGs that encircle ssDNA for conventional helicase activity. (B) Native PAGE gel time course of results using CMG + Mcm10. Lanes 1–5 show the reaction using the two-tailed substrate described in (A) while lanes 6–10 and 11–15 show control reactions using the same duplex with only a single tail at one end or the other. The migration of the substrates and unwound/trapped product are indicated to the left and right of the gel. (C) A plot of the data from (B) shows the averages of three independent trials using these substrates. The error bars show the standard deviations. See also Figure 4—figure supplements 2–3.

Figure 4—figure supplement 1. Addition of oligo trap prevents further loading of CMGM onto the two-tailed substrate.

The experiment in Figure 4B was repeated with addition of the oligo trap prior to addition of ATP/Mcm10 (left gel) or 45” after addition of ATP/Mcm10 (right gel) as in Figure 4B. The reaction scheme is shown above the gels, indicating the two different points of trap addition, and the data are plotted below. The values shown are the average of three independent experiments and the error bars show one standard deviation.

Figure 4—figure supplement 2. CMG alone does not unwind the origin-duplex substrate.

The experiment in Figure 4B, lanes 1-5, was repeated with CMG alone (no Mcm10). Because Mcm10 was not added, the experimental design was streamlined to an initial 2’ incubation with CMG, ATP, and the substrate (for CMG loading) after which the trap oligo was added to monitor product formation (scheme at top). Shown are the native PAGE gels from two separate experiments.

Figure 4—figure supplement 3. Unwinding of the origin-duplex substrate in Figure 4 requires ATP and CMG.

The experiment in Figure 4B, lanes 1-5, was repeated to demonstrate that unwinding of the two-tailed substrate by the complete system (lanes 2-6, with CMG, Mcm10 and ATP) is dependent upon both CMG (lanes 12-16, no CMG) and ATP (lanes 7-11, no ATP). This experiment also demonstrates that Mcm10 alone does not unwind the substrate (lanes 12-16). For these experiments, CMG was pre-incubated with the substrate in the presence of ATP (where indicated) for 10’ before the addition of Mcm10.