Fig. 2. RPA increases the rate of DNA unwinding by CMG.

a Fork DNA containing 236-bp dsDNA was incubated with CMG in the presence of ATPγS for binding to 3′ dT₄₀ tail. ATP was added with (lane 4) or without (lane 3) RPA and incubated further before separating on 3% agarose. DNA was labeled internally with multiple Cy5 fluorophores on both strands. This experiment was performed once. b Fork DNA substrate containing 252-bp long dsDNA, followed by 28-bp duplex and Cy5-modification on the excluded strand was bound by CMG. ATP was added to initiate translocation by the helicase. c CMG-mediated displacement of Cy5-labeled strand in the presence of RPA. When included in ATP buffer, RPA prevents reannealing of DNA behind the helicase as well as new CMG binding. d CMG-mediated displacement of Cy5-labeled strand in the absence of RPA. DNA rewinds within the 252-bp duplex region. To prevent rehybridization of the Cy5-labeled strand to long DNA substrate, excess competitor oligonucleotide containing complementary 28-nt sequence to long DNA was added with ATP. To achieve single-turnover kinetics, excess dT₄₀ oligonucleotide was included in ATP buffer that captures any free CMG. e, f Percentage of Cy5-modified strand displaced versus time by CMG in the presence e and absence of RPA f. The data in c–f represent mean from n = 2 independent experiments, and were fit to Eq. (1) with m = 1 or 2 (see Methods) resulting in best R² value. Solid lines are fits to Eqs. (2) and (3) in e and f, respectively. Source data are provided as a Source Data file.