Supporting evidence for DNA shearing as a mechanism for origin unwinding in eukaryotes

Abstract

ABSTRACTOur earlier study demonstrated that head-to-head CMGs that encircle duplex DNA and track inward at origins, melt double-strand (ds) DNA while encircling the duplex by pulling on opposite strands and shearing DNA apart (Langston and O’Donnell (2019) eLife9, e46515). We show here that increasing the methylphosphonate neutral DNA from 10 nucleotides in the previous report, to 20 nucleotides, reveals that CMG encircling duplex DNA only interacts with the tracking strand compared to the non-tracking strand. This significantly enhances support that CMG tracks on duplex DNA by binding only one strand. Furthermore, EMSA assays using AMPPNP to load CMG onto DNA shows a stoichiometry of only 2 CMGs on an origin mimic DNA, containing a 150 bp duplex with two 3 prime single-strand (ss) tails, one on each end, enabling assay of dsDNA unwinding by a shearing force produced by only two head-to-head CMGs. The use of non-hydrolysable AMPPNP enabled a preincubation for CMG binding the two 3 prime tailed origin mimic DNA, and gave robust unwinding of dsDNA by head-to-head CMG-Mcm10’s. With this precedent, it is possible to envision that the cell may utilize opposing dsDNA motors to unwind DNA for other types of DNA transactions besides origin unwinding.