DOI: 10.5061/dryad.gf1vhhmxc
title: Mechanism of BRCA1-BARD1 function in DNA end resection and DNA protection

description: DNA double-strand break (DSB) repair by homologous recombination (HR) is
 initiated by DNA end resection, a process involving the controlled
 degradation of the 5'-terminated strands at DSB sites. The breast
 cancer suppressor BRCA1-BARD1 not only promotes resection and HR, but it
 also protects DNA upon replication stress. BRCA1-BARD1 counteracts the
 anti-resection and pro-non-homologous end-joining factor 53BP1, but
 whether it functions in resection directly has been unclear. Using
 purified recombinant proteins, we show here that BRCA1-BARD1 directly
 promotes long-range DNA end resection pathways catalyzed by the EXO1 or
 DNA2 nucleases. In the DNA2-dependent pathway, BRCA1-BARD1 stimulates DNA
 unwinding by the WRN/BLM helicase. Together with MRE11-RAD50-NBS1 (MRN)
 and phosphorylated CtIP, BRCA1-BARD1 forms the BRCA1-C complex, which
 stimulates resection synergistically to an even greater extent. A mutation
 in phosphorylated CtIP (S327A), which disrupts its binding to the BRCT
 repeats of BRCA1 and hence the integrity of the BRCA1-C complex, inhibits
 resection, showing that BRCA1-C is a functionally integrated ensemble.
 While BRCA1-BARD1 stimulates resection in DSB repair, it paradoxically
 also protects replication forks from unscheduled degradation upon stress,
 which involves an HR-independent function of the recombinase RAD51. We
 show that in the presence of RAD51, BRCA1-BARD1 instead inhibits DNA
 degradation. Based on our data, the presence and local concentration of
 RAD51 might determine the balance between the pro-nuclease and the DNA
 protection functions of BRCA1-BARD1 in various physiological contexts.
description: In the uploaded files, we present mass photomether and
 single-molecule magnetic tweezer data from the main and extended data
 figures. The uploaded files include all the information required to plot
 the graphs as indicated in the attached main and extended data
 figures.  Mass photometry measurements were performed
 on a TwoMP mass photometer (Refeyn Ltd). First, borosilicate microscope
 glass plate (No. 1.5 H thickness, 24 x 50 mm, VWR) were cleaned by
 sequential soaking in Milli-Q-water, isopropanol and Milli-Q-water
 followed by drying under a stream of clean nitrogen. Next, silicone
 gaskets (CultureWell Reusable Gasket, Grace Bio-Labs) were placed on the
 clean coverslip to create a defined well for sample delivery. To convert
 optical reflection-interference contrast into a molecular mass, a known
 protein size marker (NativeMark Unstained Protein Standard, Invitrogen)
 was measured on the same day. For mass measurements, gaskets were filled
 with 18 ml measurement buffer (25 mM Tris-HCl pH 7.5, 1 mM ATP, 3 mM
 magnesium acetate) to allow focusing the microscope onto the coverslip
 surface. Subsequently, 40 nM of either BRCA1 or BRCA1-BARD1 were added
 into the well (final volume, 20 ml) and sample binding to the coverslip
 surface was monitored for 1 min using the software AcquireMP (Refeyn Ltd).
 Data analysis was performed using DiscoverMP software (Refeyn
 Ltd). Single-molecule magnetic tweezer experiments were
 carried out in a custom-built magnetic tweezers setup. The DNA constructs
 were linked at their biotinylated ends with streptavidin-coated magnetic
 beads (Dynabeads M280 [Thermo Fischer Scientific]) and flushed into the
 flow cell, where the bottom slide was coated with antidigoxigenin to
 ensure specific surface binding. Moving the magnet closer to the flow cell
 resulted in the stretching of the DNA molecules that were attached to a
 magnetic bead. Tracking of the magnetic beads for all measurements was
 conducted at 300 Hz using video microscopy and real-time GPU-accelerated
 image analysis. The magnetic forces were calibrated based on fluctuation
 analysis. The measurements were performed in a reaction buffer (25 mM
 Tris-acetate pH 7.5, 2 mM magnesium acetate, 1 mM ATP, 1 mM DTT, 0.1 mg/ml
 BSA), with the indicated protein concentrations at a temperature of 37°C
 and forces between 15-25 pN. The analysis of the recorded traces was
 conducted with a custom written MATLAB program. We considered only traces
 from measurements where the magnetic bead position was traceable for at
 least 300 s. The acquired processivity and velocity for the unwinding
 events were calculated by fitting linear segments to parts of the recorded
 traces with approximately constant velocity, which were used to construct
 the histograms and for statistical analysis. To quantify the ratio of
 rewinding/unwinding events, the total number of the two events, acquired
 as described above, was determined for a fixed period of 300 sec for each
 recorded trace. To characterize the different protein combinations and WRN
 variants, the difference between the maximum value and the minimum value
 of DNA extension for a given molecule was calculated during the first 300
 sec and expressed as ΔDNA-length. Each dot represents one measured
 molecule. All Data were named according to how they
 appear in the results section. Main Fig. 2b,c, Extended Data Fig. 2f-h,
 Extended Data Fig. 3j,  Extended Data Fig 7d are single-molecule magnetic
 tweezer data. Extended Data Fig. 1a are Mass photometry data.
description: # Mechanism of BRCA1-BARD1 function in DNA end resection and DNA
 protection Here, we present mass photometer data (.mp and .mp4 files) and
 single-molecule magnetic tweezer data (.dat files) from the main and
 extended data figures of the manuscript, " Mechanism of BRCA1-BARD1
 function in DNA end resection and DNA protection". The files uploaded
 include all the information needed to plot the panels as indicated in the
 attached main and extended data 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
 presented in the study's main and supplementary figures.  **Mass
 photometer data** We shared the movies where molecules from the indicated
 sample are visualized based on their optical reflection-interference
 contrast as black objects with white edges. Each movie was recorded for
 one minute and analyzed with DiscoverMP to obtain the counts plotted in
 the images.  **Single-molecule Magnetic Tweezer data** We shared the raw
 numbers of the measured traces for the indicated sample. They correspond
 to the unwound DNA (named as DNA length and measured in bp) during the
 indicated time (measured in sec).  ## Additional Information * Mass
 photometry measurements were performed on a 2MP-0132 mass photometer
 (Refeyn). The sample was loaded onto a coverslip surface and monitored by
 recording a movie recording for 1 minute using AcquireMP (Refeyn Ltd)
 software and analysed 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 flap 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. The submitted files can be opened using
 Excel.  * All the data are named according to their appearance in the
 manuscript's results section, with the corresponding figure number. 
 ## 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 and
 plotting of magnetic tweezer data was carried out in Origin Lab 2019.
 DiscoverMP (Refeyn Ltd, version v2023 Rl.2) was used for mass photometry
 data analysis.