HROB promotes the MCM8-9 helicase in DNA damage response. To understand how HROB activates MCM8-9, we defined their interaction interface. We showed that HROB makes important yet transient contacts with both MCM8 and MCM9, and binds the MCM8-9 heterodimer with the highest affinity. MCM8-9-HROB prefer branched DNA structures, and display low DNA unwinding processivity. MCM8-9 unwinds DNA as a hexamer that assembles from dimers on DNA in the presence of ATP. The hexamer involves two repeating protein-protein interfaces between the alternating MCM8 and MCM9 subunits. One of these interfaces is quite stable and forms an obligate heterodimer across which HROB binds. The other interface is labile and mediates hexamer assembly, independently of HROB. The ATPase site formed at the labile interface contributes disproportionally more to DNA unwinding than that at the stable interface. Here, we show that HROB promotes DNA unwinding downstream of MCM8-9 loading and ring formation on ssDNA.

Here, we present mass photometer data and single-molecule magnetic tweezer data from the main and supplementary figures of the manuscript, "Mechanism of DNA unwinding by MCM8-9 in complex with HROB". The files uploaded include all the information needed to plot the panels as indicated in the attached main and supplementary figures and the file names.

We share information obtained through mass photometry movies and single-molecule magnetic tweezers techniques to measure DNA unwinding, featured in both the main and supplementary figures of the study, "Mechanism of DNA unwinding by MCM8-9 in complex with HROB".

Mass photometry measurements were performed on a 2MP-0132 mass photometer (Refeyn). Sample binding to the coverslip surface was monitored by recording a movie for 1 minute using AcquireMP (Refeyn Ltd) software. Data analysis was performed using DiscoverMP (Refeyn Ltd). For each peak analyzed, at least 500 events were considered. To convert the measured optical reflection-interference contrast into a molecular mass, a known protein size marker (NativeMark^TM Unstained Protein Standard, Invitrogen) was measured. The raw data from the mass photometer are presented as .mp files. These files follow the open HDF5 file format and can be read with any software that supports HDF5. However, to enable the reader to reproduce the results from the MP files, file should be opened in DiscoverMP. In addition, if one right-clicks on the movie and selects Export > Movie on the DiscoverMP software (Refeyn Ltd, version v2023 Rl.2), it will be saved as a.mp4, which can be played with Windows media player.

Single-molecule magnetic tweezers were performed using a Holliday Junction DNA conctruct to measure DNA unwinding. Magnetic tweezers single molecule data were collected using Labview 2016 and CUDA software as published (Huhle et al, Nat Commun, 2015). Data analysis as well as plotting of magnetic tweezers data was carried out in Origin Lab 2019. 

All the data are named according to how they appear in the results section of the manuscript with the corresponding figure number. Main Figure 1a-c, 4h, 5b, 6c and Supplementary Figure 1b, 7a, 7b, 7h, 7i and 9c-e are mass photometry data. Main figure 3c-e and supplementary figure 5c-e are single molecule data.