DOI: 10.5061/dryad.mw6m9069c
title: Data from: RAD51C-XRCC3 complex regulates FANCM-mediated R-loop resolution to safeguard genome integrity

description: Fanconi anemia (FA) is characterized by bone marrow failure, congenital
 abnormalities, and cancer predisposition. Mutations in RAD51 paralogs have
 been identified in FA-like disorders and cancers. Although the role of
 RAD51 paralogs is well established in homologous recombination
 (HR)-mediated DNA repair, little is known about their role during
 replication stress responses. Here, we report that the RAD51C-XRCC3 (CX3)
 complex of RAD51 paralogs participates in the FA-pathway of R-loop
 tolerance mechanism. CX3 complex suppresses R-loops,
 transcription-replication collisions (TRC), and associated genome
 instability in physiological and replication stress conditions.
 Mechanistically, the CX3 complex physically interacts with FANCM and
 facilitates its recruitment to the R-loop sites to promote its resolution.
 Notably, cells expressing RAD51C R258H pathological mutant exhibit
 defective interaction with FANCM and display inefficient R-loop
 processing. Strikingly, the R-loop resolution function of RAD51C is
 independent of its ATPase activity, which is required for HR, fork
 protection, and restart. Collectively, our work identifies a novel role of
 the CX3 complex in preventing R-loop-induced genome instability by
 regulating FANCM-mediated R-loop resolution.
description: # Data from: RAD51C-XRCC3 complex regulates FANCM-mediated R-loop
 resolution to safeguard genome integrity Dataset DOI:
 [10.5061/dryad.mw6m9069c](https://doi.org/10.5061/dryad.mw6m9069c) ##
 Description of the data and file structure All the data, including
 representative microscopic images, Western blot images, quantification
 Excel files, and AlphaFold3 structural data, are included in the submitted
 files. **File Structure:**\ Organized by Figures and Analysis Fig. 1 \|-
 1A \|- |- 1A data images/ excel file/ blot pdf \|- 1B ... ### Files and
 variables **Data processing method:** For S9.6 quantification experiments:
 Raw nuclear signal intensities for S9.6 were obtained from images of
 around &gt; 80 cells per sample using ImageJ software. DAPI signal was
 used as a mask to get nuclear boundary. The mean intensity value for the
 control sample was calculated. Intensity of all nuclei from all the
 samples were divided by the mean intensity of the control sample to obtain
 the "relative S9.6 nuclear intensity" values. The "relative
 S9.6 nuclear intensity" values for all collected cells were plotted
 as y-axis of the box-whisker plot against the samples analyzed in the
 x-axis. For box plots, boxes and whiskers indicate the 25th to the 75th
 and the 10th to the 90th percentiles, respectively, and median values are
 indicated. Statistical tests were performed on mean values of three or
 more biological replicates. *P* values and the statistical test applied
 are described in the "analysis" section (sheet) of the Excel
 file. R1, R2 and R3 sheets in the Excel data files denote the 3 biological
 repeats.  For other IF foci count experiments: Nuclear foci were counted
 by applying DAPI signal as mask using the ImageJ software by setting an
 appropriate "Find Maxima" threshhold value (Threshhold remains
 same across all samples in a given experiment). As the maxima readout
 comes in the multiple of 255, the raw foci count numbers were divided by
 255 to get the actual foci fount per cell. "Foci count/cell"
 were plotted as y-axis of the scatter plot against the sample name in the
 x-axis. Statistical tests were performed on mean values of three or more
 biological replicates. *P* values and the statistical test applied are
 described in the "analysis" section (sheet) of the Excel file.
 R1, R2, and R3 sheets in the Excel data files denote the 3 biological
 repeats.  **Abbreviations:** The following abbreviations may be used in
 some of the files for different samples as a short-hand. | Sample Name |
 Abbreviations | | :---------- | :------------------- | | shControl |
 shCtrl | | shXRCC3 | shX3 | | shRAD51C | shC / sh51C / shR51C | | shXRCC2
 | shX2 | | shFANCD2 | shFD2 | | shFANCM | shFM | #### File: Fig._1.zip
 **Description:** Data corresponding to Main Figure 1 is present in this
 folder. #### File: Fig._2.zip **Description:** Data corresponding to Main
 Figure 2 is present in this folder. #### File: Fig._3.zip **Description:**
 Data corresponding to Main Figure 3 is present in this folder. #### File:
 Fig._4.zip **Description:** Data corresponding to Main Figure 4 is present
 in this folder. #### File: Fig._5.zip **Description:** Data corresponding
 to Main Figure 5 is present in this folder. #### File: Fig._6.zip
 **Description:** Data corresponding to Main Figure 6 is present in this
 folder. #### File: Fig._7.zip **Description:** Data corresponding to Main
 Figure 7 is present in this folder. #### File: Fig._S1.zip
 **Description:** Data corresponding to Supplementary Figure 1 is present
 in this folder. #### File: Fig._S2.zip **Description:** Data corresponding
 to Supplementary Figure 2 is present in this folder. #### File:
 Fig._S3.zip **Description:** Data corresponding to Supplementary Figure 3
 is present in this folder. #### File: Fig._S4.zip **Description:** Data
 corresponding to Supplementary Figure 4 is present in this folder. ####
 File: Fig._S5.zip **Description:** Data corresponding to Supplementary
 Figure 5 is present in this folder. #### File: Fig._S6.zip
 **Description:** Data corresponding to Supplementary Figure 6 is present
 in this folder. #### File: Fig._S8.zip **Description:** Data corresponding
 to Supplementary Figure 8 is present in this folder. #### File:
 Fig._S10.zip **Description:** Data corresponding to Supplementary Figure
 10 is present in this folder. #### File: Alphafold3_Results.zip
 **Description:** Alphafold modeling data with Figure 5F and 5G, S7 and S9
 are present in this folder. The folder contains only selected (model 1)
 AF3-generated model for WT and R258H RAD51C-XRCC3 complex interaction with
 FANCM. All the other predicted models of AlphFold3 (for Figs. S7, S9 and
 S11), including those with the WT CX3 and R258H mutant with FANCM, can be
 accessed from Model Archive with accession IDs ma-xdl20 and ma-miymb.