Fig. 2: MCM8-9 with HROB unwinds branched DNA structures.

a DNA unwinding by MCM8-9 without or with HROB and RPA, as indicated, with Y and Holliday junction (HJ) DNA substrates with 1 mM ATP, 5 mM magnesium acetate and 15 mM NaCl. Red asterisk, radioactive label. Representative of n = 3 independent assays is shown. b Quantification of helicase assays, such as shown in panel a and Supplementary Fig. 4a. Error bars, SEM; n = 3 independent experiments. c Quantification of DNA binding assays with MCM8-9, such as shown in Supplementary Fig. 4b. Error bars, SEM; n = 3 independent experiments. d Quantification of DNA binding assays with HROB such as shown in Supplementary Fig. 4b. Error bars, SEM; n = 3 independent experiments. e A cartoon representing two possible translocation polarities of MCM8-9 on M13-based substrates used in panel f. See text for details. f DNA unwinding by MCM8-9 with/without HROB, using M13-based substrates as indicated, and 30 mM NaCl. Top, quantification; error bars, SEM; bottom, representative of n = 3 independent experiments, except n = 4 independent experiments for no overhang substrate. Red asterisk, radioactive label. g Quantification of ATP hydrolysis (expressed as arbitrary units, arb. units, normalized to wild type MCM8-9 alone as 1) by 200 nM wild type MCM8-9 and ATP-binding deficient mutant MCM8 (K460A)-9 (K358A) with or without HROB. 7.2 μM (in nucleotides) M13-based circular ssDNA substrate was used as a co-factor. Error bars, SEM; n = 3 independent experiments. h ATP hydrolysis, expressed in % of total ATP hydrolysed by MCM8-9 with and without 100 nM HROB in the presence of various oligonucleotide-based DNA substrates (7.2 μM, in nucleotides) and RPA (0.58 μM). Bars show range; n = 2 independent experiments. i Relationship between ATP hydrolysis by MCM8-9 (200 nM) and the concentration of DNA (μM, in nucleotides) without and with HROB (100 nM). V is the rate of ATP hydrolysis. Error bars, SEM; n = 4 independent experiments for [ssDNA] ranging from 0.225 to 3.6 μM, n = 7 for [ssDNA] ranging from 7.2 to 14.4 μM and n = 5 independent experiments for [ssDNA] ranging from 28.8 to 115.2 μM.