Mechanism of BRCA1-BARD1 function in DNA end resection and DNA protection

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.

Methods
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.