Harmful R-loops are prevented via different cell cycle-specific mechanisms

Identifying how R-loops are generated is crucial to know how transcription compromises genome integrity. We show by genome-wide analysis of conditional yeast mutants that the THO transcription complex, prevents R-loop formation in G1 and S-phase, whereas the Sen1 DNA-RNA helicase prevents them only in S-phase. Interestingly, damage accumulates asymmetrically downstream of the replication fork in sen1 cells but symmetrically in the hpr1 THO mutant. Our results indicate that: R-loops form co-transcriptionally independently of DNA replication; that THO is a general and cell-cycle independent safeguard against R-loops, and that Sen1, in contrast to previously believed, is an S-phase-specific R-loop resolvase. These conclusions have important implications for the mechanism of R-loop formation and the role of other factors reported to affect on R-loop homeostasis.

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Policy information about availability of computer code Data collection Microscopy images were acquired the with a Leica DM6000 microscope equipped with a DFC390 camera and LAS AX v2.0 image acquisition software (Leica). Real-time quantitative PCRs (qPCRs) were performed on a 7500 Fast Real-Time PCR system (Applied Biosystems, Carlsbad, CA). Quantitative PCR results were obtained from a 7500 FAST Real-Time PCR System equipped with 7500 Software v2.3. Flow cytometry analysis were performed using a BFACScalibur (Becton Dickison fluorescence-activated cell analyzer) and analyzed by BD CellQuest Pro Software v5.1.

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Western blots bands were quantified using ImageStudio software v3.1 (LI-COR biosciences). Display of plots and statistical analyses were carried out using the Prism software (GraphPad) v8. Images were processed using Adobe Photoshop CS4. For genome wide data, sequenced paired-ends reads were subjected to quality control pipeline using the FASTQ Toolkit V.1.0.0 software (Illumina) and then mapped to the Saccharomyces cerevisiae reference genome using the Rsubread V 2.0.1 software package. Mapped reads were assigned to Watson or Crick strand using SAMtools V 1.1072. Peak calling was performed with chromstaR V 1.12.0 software package43. For comparative analysis, regions covered by peaks in the two conditions that are being compared were merged and fused when closer than 200 bp distance using BEDtools V 2.27.173. The differential enrichment of these regions in each condition was performed using csaw V 1.20.0 software package74. After that, edgeR package (v3.20.9) was used in order to calculate log2FC and p-value of the peaks. Coverage profiling were obtained using bamCoverage tool from deepTools V 3.4.378. Genome example regions were plotted using IGV V 2.8.2 software79.
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April 2020

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Methodology Sample preparation
For FACS analysis, each yeast culture was centrifuged and washed with sodium citrate 50 mM pH 7.5 and resuspended in 1 ml of ethanol 70%. The samples could be stored at 4ºC until processing. Cells were washed once with sodium citrate 50 mM pH 7.5 and incubated 1 hour at 50ºC with 25 μl RNase A (10mg/ml). 50 μl of 20 mg/ml proteinase K was added and samples were incubated 1 hour at 50ºC. 1 ml of sodium citrate containing 16 μg/ml propidium iodide was added and samples were sonicated until cells were completely disaggregated. The samples were stored 30 minutes in the dark or 12-48 hours at 4ºC before the analysis. Before the flow cytometry, cells were sonicated 5 seconds at 10% amplitude and scored in FACScalibur (Becton Dickison fluorescence-activated cell analyzer). For each histogram 100000 yeast cells were analyzed.