Fig. 1. HROB interacts with both subunits of the MCM8–9 heterodimer.

a. A schematic representation of HROB and HROB-ΔOB, an internal deletion mutant of HROB lacking the OB-fold domain.

b. Purified wild type human FLAG-HROB and FLAG-HROB-ΔOB used in this study.

c. Quantification of assays such as shown in panel Extended Data Fig. 1a (dashed: HROB-ΔOB, solid: HROB). Error bars, SEM; n = 3.

d. Purified wild type human MBP-MCM9 and FLAG-MCM8 (MCM8–9 complex) used in this study.

e. DNA unwinding by MCM8–9 (100 nM) and either HROB or HROB-ΔOB (both 50 nM), using M13-based circular DNA substrate with 30 mM NaCl. The red asterisk indicates the position of the radioactive label. Top, quantitation; error bars, SEM; n = 3; bottom, a representative experiment.

f. A schematic representation of human HROB binding to human MCM8–9 hexameric complex modelled using AlphaFold2.

g. A schematic representation of the interaction between human MCM9 and HROB. Highlighted are the residues HROB-F553 and MCM9-M45 that interact with each other. F553E and M45E point mutations in HROB and MCM9, respectively, were tested in further assays.

h. A schematic representation of the interaction between human MCM8 and HROB. Highlighted are the residues HROB-L405 and MCM8-L387 that interact with each other. The L405D and L387E point mutations were tested in further assays.

i. MCM9-M45E mutation disrupts MCM9-HROB interaction in cell extracts. Lysates from HEK293T cells expressing GFP, HA-MCM9-WT or HA-MCM9-M45E were subjected to HA-immunoprecipitation. Immunoblotting of HROB, MCM8 and HA are presented along with a loading control, vinculin. Shown is a representative of two independent experiments.

j. DNA unwinding with indicated mutants (100 nM) to test the impact of disrupted HROB (50 nM) interaction with MCM9 using M13-based circular DNA substrate with 30 mM NaCl. HROB wild type with MCM8–9 wild type is replotted as in panel e for reference. The red asterisk indicates the position of the radioactive label. Top, quantitation; error bars, SEM; n = 3; bottom, a representative experiment.

k. MCM8-L387E mutation disrupts MCM8-HROB interaction. Lysates from HEK293T cells expressing GFP, HA-MCM8-WT and HA-MCM8-L387E were subject to HA-immunoprecipitation. Immunoblotting of HROB, MCM9 and HA are presented along with a loading control, vinculin. Shown is a representative of two independent experiments.

l. DNA unwinding with indicated mutants to test the impact of disrupted HROB (25 nM) interaction with MCM8 (50 nM) using M13-based circular DNA substrate with 150 mM NaCl. The red asterisk indicates the position of the radioactive label. Top, quantitation; error bars, SEM; n = 3; bottom, a representative experiment.