Fig. 3. Single-molecule analysis of the MCM8-9 helicase with HROB.

a Quantitation of gel-based assays showing DNA unwinding by MCM8-9 (100 nM) and HROB (33 nM), using substrates with different lengths of duplex DNA (overhang length 20 nt) as indicated, with 14 mM NaCl. Red asterisk, radioactive label. Error bars, SEM; n = 3 independent experiments. b Schematic representation of the magnetic tweezer setup and the investigated Holliday junction construct with 262 bp in each arm. When the protein ensemble is added, MCM8-9 with HROB can translocate in the direction of the arms, causing the measured DNA length to increase. c Activity of MCM8-9 and HROB with ATP, as indicated. Successive DNA unwinding events (highlighted by an asterisk), resulting in a net-increase of DNA length, were observed. d Magnified example trace for a representative unwinding event (highlighted by an asterisk) by MCM8-9 and HROB. e MCM8-9 with HROB do not unwind DNA with nonhydrolysable ATP-γ-S instead of ATP. f. Probability distribution of DNA unwinding processivity by MCM8-9 with HROB, with a mean of 41 ± 5 bp, of events leading to DNA extension. Error, 2SEM; DNA unwinding of 32 molecules was measured. g Probability distribution of DNA unwinding velocity by MCM8-9 with HROB, with a mean of 10 ± 3 bp sec⁻¹, of events leading to DNA extension. Error, 2SEM; DNA unwinding of 32 molecules was measured.