Fig. 1.

Purified recombinant DmCMG, bulk unwinding, single-molecule assay principle, and observed single-molecule DNA unwinding trajectories. a Coomassie stained 4–12% SDS-PAGE gel of purified DmCMG. Sld5, Psf1-3 forms GINS. b Example of bulk unwinding of duplex DNA, radiolabelled at 5′ ends, by CMG. The 60 bp duplex with 40 nt 3′ polyT tail is unwound into the two single strands. c To form the single-molecule assay a single 2.7 kilobase dsDNA molecule, with a polyT 3′ overhang to serve as a high affinity binding site for CMG, is tethered at one end to a PEGylated glass surface via biotin and streptavidin binding; and to a super-paramagnetic microsphere at the other end via digoxigenin/anti-digoxigenin binding. A force, F, is applied using a pair of neodymium cube magnets. Precise tracking of the microsphere vertical displacement gives the extension of each single DNA molecule. d Protocol for experiment. Surface-tethered DNA is incubated with CMG in the presence of ATPγS to aid helicase loading onto the 3′ ssDNA tail. ATP is then introduced through a 4–5 sample chamber volume wash, and unwinding begins, with data being collected for at least 90 min. e Typical examples of single-molecule DNA unwinding by CMG at 20pN and 4 mM ATP, filtered to 0.17 Hz using a mean running window. CMG is a slow helicase, acting non-monotonically with heterogeneity both within and between individual enzyme unwinding events. f Single-molecule unwinding CMG trajectories exhibit not only unwinding but also, what can be crudely described as, pause-like dynamics and annealing, which we attribute to reverse motion of the helicase. Example regions have been manually highlighted