The Smc5/6 complex counteracts R-loop formation at highly transcribed genes in cooperation with RNase H2

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 ..., , , # 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-DN...