DOI: 10.5061/dryad.wdbrv15wq
title: Mechanism of DNA unwinding by MCM8-9 in complex with HROB

description: 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.
description: 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<sup>TM</sup> 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 &gt; 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.  
description: # Mechanism of DNA unwinding by MCM8-9 in complex with HROB
 [https://doi.org/10.5061/dryad.wdbrv15wq](https://doi.org/10.5061/dryad.wdbrv15wq) 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. ## Description of the data and file structure We share information obtained through mass photometry and single-molecule magnetic tweezers techniques to measure DNA unwinding, featured in the study's main and supplementary figures. ## Sharing/Access information Data was derived from the following sources: * 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). To convert the measured optical reflection-interference contrast into a molecular mass, a known protein size marker (NativeMarkTM 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, the files should be opened in DiscoverMP. In addition, if one right-clicks on the movie and selects Export &gt; Movie on the DiscoverMP software (Refeyn Ltd, version v2023 Rl.2), it will be saved as a .mp4 file which is also provided for all the figures. * Single-molecule magnetic tweezers were performed using a Holliday Junction DNA construct to measure DNA unwinding. The proteins were pre-mixed and then flushed into the flow cell containing the DNA construct, and the traces were recorded subsequently. Magnetic tweezers based-single molecule data were collected using Labview 2016 and CUDA software as published (Huhle et al, Nat Commun, 2015). Data analysis and plotting of magnetic tweezer data was conducted in Origin Lab 2019.  * All the data are named according to their appearance in the manuscript's results section, with the corresponding figure number. Main Figures 1a-c, 4h, 5b, 6c, and Supplementary Figures 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.   ## Code/Software Data collection Magnetic tweezers single molecule data were collected using Labview 2016 and CUDA software as published (Huhle et al, Nat Commun, 2015). AcquireMP (Refeyn Ltd, version 2023 Rl.l) was used for mass photometry data collection. Data analysis Data analysis as well as plotting of magnetic tweezers data was carried out in Origin Lab 2019. DiscoverMP (Refeyn Ltd, version v2023 Rl.2) was used for mass photometry data analysis.