DOI: 10.5061/dryad.3xsj3txpg
title: The Smc5/6 complex counteracts R-loop formation at highly transcribed genes in cooperation with RNase H2

description: The R-loop is a common transcriptional by-product that consists of an
 RNA-DNA duplex joined to a displaced strand of genomic DNA. While the
 effects of R-loops on health and disease are well established, there is
 still an incomplete understanding of the cellular processes responsible
 for their removal from eukaryotic genomes. Here, we show that a core
 regulator of chromosome architecture —the Smc5/6 complex— plays a crucial
 role in the degradation of R-loop structures formed during gene
 transcription. Consistent with this, mutants defective in the Smc5/6
 complex and enzymes involved in R-loop resolution show strong synthetic
 interactions and accumulate high levels of RNA-DNA hybrid structures in
 their chromosomes. Importantly, we demonstrate that the Smc5/6 complex
 recognizes specific types of RNA-DNA hybrid structures in vivo and
 promotes the degradation of R-loops by RNase H enzymes. Collectively, our
 results reveal a crucial role for the Smc5/6 complex in the removal of
 toxic R-loops formed at highly transcribed genes and telomeres.
description: # The Smc5/6 complex counteracts R-loop formation at highly transcribed
 genes in cooperation with RNase H2
 [https://doi.org/10.5061/dryad.3xsj3txpg](https://doi.org/10.5061/dryad.3xsj3txpg) ## Description of the data and file structure **All files in .csv format represents quantitative data (raw values).** ### Folder - Figure 2-source data #### Figure 2-source data 1 (graph in Figure 2 Panel A in manuscript): Quantification of RNA-DNA hybrid foci in chromatin spread shown in the top part of Figure 2 Panel A (in the top part only a representative field of view is shown - the data is derived from taking an average of at least 15-20 of such fields of view). Images were acquired using Nikon Eclipse Ti2 inverted microscopy with an oil immersion 100 x objective. Fraction of foci (number of foci/nucleus) is shown for each genetic mutant of *S. cerevisiae*. 100-200 nuclei were visualized (blue) and manually counted for each replicate to obtain the fraction of nuclei with detectable RNA-DNA hybrids (orange).  **Figure 2-source data 1-strain info.csv** : identity/genotype of genetic mutants assayed. **Figure 2-source data 1-quant.csv** : fraction of foci for three independent replicates for all genetic mutants. Only column "Nuclei more than 10 foci" is used for downstream analyses. For each replicate (n&gt;3), about 150 nuclei were visualized and manually counted to obtain the fraction with detectable RNA-DNA hybrid foci using the 3D measurement module of the NIS-Elements software (Nikon Instruments Inc). **Figure 2-source data 1-stats.csv** : the output of statistical tests (t-test) performed with fraction of foci as dependent and mutant as independent variable. #### Figure 2-Source data 2 (graph in Figure 2 Panel B in manuscript): Quantification of RNA-DNA hybrid foci by chromatin spread with or without RNase H1 over-expression in the top part of Figure 2 Panel B (in the top part only a representative field of view is shown - the data is derived from taking an average of at least 15-20 of such fields of view). Images were acquired using Nikon Eclipse Ti2 inverted microscopy with an oil immersion 100 x objective. Fraction of foci (number of foci/nucleus)  is shown for each genetic mutant of *S. cerevisiae*. 100-200 nuclei were visualized (blue) and manually counted for each replicate to obtain the fraction of nuclei with detectable RNA-DNA hybrids (orange).  **Figure 2-Source data 2-sheet 1-quant.csv** :  fraction of foci for three independent replicates for all genetic mutants. For each replicate (n&gt;3), about 150 nuclei were visualized and manually counted to obtain the fraction with detectable RNA-DNA hybrid foci using the 3D measurement module of the NIS-Elements software (Nikon Instruments Inc). **Figure 2-Source data 2-sheet 1-stats.csv** : the output of statistical tests (t-test) performed with fraction of foci as dependent and mutant as independent variable.  ### Folder - Figure 3-Source data #### Figure 3-Source data 1 (graph in Figure 3 Panel A in manuscript): Quantification of q-PCR analyses following immuno-precipitation of RNA-DNA hybrids. **Figure 3-Source data 1-strain info.csv** : identity/genotype of genetic mutants assayed.  **Figure 3-Source data 1-quant.csv** : the raw and normalized percent input obtained from at least three independent replicate q-PCR assays at three genetic locations for all strains studied. The "input" column is the data that was obtained from the CFX Maestro Bio-Rad software after performing the q-PCR assay and the relative abundance of RNA-DNA hybrid immunoprecipitated in each region was normalized to the signal obtained in the inputs from the wild type (7528) for each gene (18S, 21S and Telo).  **Figure 3-Source data 1-stats.csv** : t-test results from the pairwise comparison between the strains studied using normalized percent input as dependent and mutants as independent variable. #### Figure 3-Source data 2 (graph in Figure 3 Panel B in manuscript): Quantification of Rad52 foci in *S. cerevisiae* genetic mutants. About 100 cells were visualized and manually counted for each replicate to obtain the fraction of cells with detectable Rad52-GFP foci (green). **Figure 3-Source data 2-strain info.csv** : identity/genotype of genetic mutants assayed. **Figure 3-Source data 2-quant.csv** : For DAPI staining and Rad52-GFP visualization, images were acquired using Nikon Eclipse Ti2 inverted microscopy with an oil immersion 100 x objective. For each replicate (n=3), about 100 cells were visualized and manually counted to obtain the number of cells with detectable Rad52 foci (green). Only the column "Percent_foci" is used for downstream analyses.  **Figure 3-Source data 2-stats.csv** : the output of the statistical test (Welch's t-test) performed between mutant strains of interest (please note column H and column G in these cells do not indicate this sheet but columns in GraphPad Prism worksheet) with percent foci as dependent and mutants as independent variables. ### Folder - Figure 5 Source data (Figure 5 and Figure 5- supplement 1 and 2 in manuscript) Raw uncropped and unedited pictures of gels to show image integrity. **Please consult figures/figure legends from the manuscript as reference for more information.** *.tiff files can be opened by any traditional image viewer. **Figure 5- Source data 1.tiff** Raw uncut and unedited gel as represented/annotated on Figure 5- Source data 3.pdf- Panel C (on left). **Figure 5-Source Data 2.tiff** Raw uncut and unedited gel as represented/annotated on Figure 5- Source data 3.pdf- Panel D (on right). **Figure 5- Source data 3.pdf** Raw uncropped gels with the relevant bands labelled for Figure 5C-D. The gels are annotated exactly as they appear on Figure 5C and 5D in the manuscript. **Figure 5-Supplement 1-Source Data 1.pdf** Raw uncropped gels with the relevant band labelled for Figure 5-Supplement 1A-B. The gels are annotated exactly as they appear on Figure 5-Supplement 1A-B in the manuscript. **Figure 5-Supplement 1-Source Data 2.tiff** Raw uncut and unedited gel as represented/annotated on Figure 5-Supplement 1-Source Data 1.pdf Panel A (on top). **Figure 5-Supplement 1-Source Data 3.tiff** Raw uncut and unedited gel as represented/annotated on Figure 5-Supplement 1-Source Data 1.pdf Panel B (at bottom). **Figure 5-Supplement 2-Source Data 1.pdf** Raw uncropped gels with the relevant band labelled for Figure 5-Supplement 2C-F. The gels are annotated exactly as they appear on Figure 5-Supplement 2C-F in the manuscript.  **Figure 5-Supplement 2-Source Data 2.tiff** Raw uncut and unedited gel as represented/annotated on Figure 5-Supplement 2-Source Data 1.pdf Panel C (top left). **Figure 5-Supplement 2-Source Data 3.tiff** Raw uncut and unedited gel as represented/annotated on Figure 5-Supplement 2-Source Data 1.pdf Panel D (top right). **Figure 5-Supplement 2-Source Data 4.tiff** Raw uncut and unedited gel as represented/annotated on Figure 5-Supplement 2-Source Data 1.pdf Panel E (bottom left). **Figure 5-Supplement 2-Source Data 5.tiff** Raw uncut and unedited gel as represented/annotated on Figure 5-Supplement 2-Source Data 1.pdf Panel F (bottom right). ### Folder - Figure 6 Source data (Figure 6C and Figure 6 -supplement 1 and 2 in manuscript) Data for Figure 6C and raw unedited pictures of gels to show image integrity. **Please consult figures/figure legends from the manuscript as reference for more information.**   **Figure 6 Source data 1.tif** Raw uncut and unedited gel as represented/annotated on Figure 6 Source data 2.pdf. **Figure 6 Source data 2.pdf** Raw uncropped gels with the relevant band labelled for Figure 6C. The gel is annotated exactly as it appears on Figure 6C in the manuscript.  **Figure 6-Source data 3-quant.csv** : Raw values of R-loop degradation from the image analysis of R-loop degradation assay as shown in Figure 6 source data 2.pdf. "Probe" is the band intensity of the bands of undegraded product (running at the top of the gel), measured using the histogram function of ImageJ software. "Background" is the intensity of the background around the band. "Probe inverted" is the difference between "Probe" and "Background" (i.e. Probe-Background). "Degraded product" is the intensity of the bands of the degraded product (running at the bottom of the gel). "Background" is the intensity of the background around the band. "DP inverted" is the difference between "Degraded product" and "Background" (i.e. Degraded product-Background). "Middle well" is the intensity of the bands of the partially-degraded product (running at the middle of the gel). "Background" is the intensity of the background around the band. "Middlewell inverted" is the difference between "Middle well" and "Background" (i.e. Middle well-Background). "Total product" is the total of "Probe inverted", "DP inverted" and "Middlewell inverted" columns. "Fraction inverted" is the fraction of "DP inverted" relative to "Total product". "Percent degraded product" is "Fraction inverted" multiplied by 100. Only the column "percent degraded product" is used for plotting and analyses (shown in the graph on the right hand side of the Figure 6C). **Figure 6-Supplement 1-Source Data 1.tif** Raw uncut and unedited gel as represented/annotated on Figure 6-Supplement 1-Source Data 3.pdf Panel A (left) and Panel B (right). **Figure 6-Supplement 1-Source Data 2.tif** Raw uncut and unedited gel as represented/annotated on Figure 6-Supplement 1-Source Data 3.pdf Panel C (bottom). **Figure 6-Supplement 1-Source Data 3.pdf** Raw uncropped gels with the relevant band labelled for Figure 6-Supplement 1A, B and C. The gels are annotated exactly as they appear on Figure 6-Supplement 1A-C in the manuscript. **Figure 6-Supplement 2-Source Data 1.pdf** Raw uncropped gels with the relevant band labelled for Figure 6-Supplement 2 A-B. The gels are annotated exactly as they appear on Figure 6-Supplement 2A-B in the manuscript. **Figure 6-Supplement 2-Source Data 2.tiff** Raw uncut and unedited gel as represented/annotated on Figure 6-Supplement 2-Source Data 1.pdf Panel A (top). **Figure 6-Supplement 2-Source Data 3.tif** Raw uncut and unedited gel as represented/annotated on Figure 6-Supplement 2-Source Data 1.pdf Panel B (bottom gels). **Figure 6-Supplement 2-Source Data 4.tif** Raw uncut and unedited gel as represented/annotated on Figure 6-Supplement 2-Source Data 1.pdf Panel B (upper gel). **All files in .tiff/.pdf format represents raw images of blots. These files can be opened by traditional image viewers or pdf viewers. For corresponding labels see figures/figure legends in the manuscript.**