Fig. 3. Single molecule analysis of the MCM8–9 helicase with HROB.
a. Quantitation of gel-based assay 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. The red asterisk indicates the position of the radioactive label. Error bars, SEM; n = 3.
b. A 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 *), resulting in a net-increase of DNA length, were observed.
d. Magnified exemplary trace for an unwinding event by MCM8–9 and HROB.
e. MCM8–9 with HROB do not unwind DNA with non-hydrolysable 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, n = 32.
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, n = 32.