Journal: Molecular cell

Article Title: Mitotic DNA synthesis is caused by transcription-replication conflicts in BRCA2-deficient cells.

doi: 10.1016/j.molcel.2022.07.011

Figure Lengend Snippet: Figure 5. MiDAS events induced by BRCA2 inactivation map to regions of R-loop formation (A) Representative genomic regions of DRIP-seq and qDRIP-seq analyses performed in asynchronous HeLa cells (Crossley et al., 2020; Hamperl et al., 2017), RFD analysis performed in HeLa cells (Petryk et al., 2016), and EU-seq and MiDAS-seq analysis performed in BRCA2-deficient (BRCA2) H1299 cells. EU-seq was performed 100 min after release from thymidine block. (B) Average qDRIP-seq signal centered across MiDAS peaks identified in BRCA2-deficient (n = 150; BRCA2; pink) or aphidicolin-treated BRCA2-proficient (n = 346; +BRCA2 +APH; blue) H1299 cells. Span of genomic region, 300 kb. (C) Transcript levels measured by EU-seq 100 min after release from single thymidine block in BRCA2-deficient (BRCA2) H1299 cells for genes found in the vicinity (±50 kb) of MiDAS sites identified in BRCA2-deficient (BRCA2) cells or at 150 randomly selected early-replicating, R-loop-prone sites. (D) G-quadruplex (G4) density at MiDAS sites identified in BRCA2-deficient (BRCA2) cells or at 150 randomly selected early-replicating, R-loop-prone sites. (E) Quantification of RNA-DNA hybrids measured by QIBC of mean chromatin-bound GFP-RNaseH1D210N signal. Graph shows the median values obtained for each of the indicated cell-cycle stages and is representative of n = 3 independent experiments. (F) Quantification of mitotic EdU foci in HeLa expressing a DOX-inducible, FLAG-tagged RNaseH1 (RNH1-FLAG), treated as indicated and collected 9 h after release from single thymidine block with 20 mM EdU added during the final hour. Graph and error bars represent the mean and SEM of a total of 156 mitotic cells per conditions from n = 3 independent experiments. Representative images are shown. Scale bars, 16 mm. (G) Replication fork directionality (RFD) measured by OK-seq (Petryk et al., 2016) at MiDAS sites identified in BRCA2-deficient H1299 cells, which contain a single gene. 22 genes transcribed in the forward direction (green) and 32 genes transcribed in the reverse direction (red) were analyzed. Span of genomic region, 150 kb. p values were calculated using an unpaired two-tailed t test (C and D) or using one-way ANOVA followed by a Tukey test (F). **p % 0.01; ****p % 0.0001. See also Figures S4 and S5 and Tables S3 and S4.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER ProLong Gold Antifade Mountant ThermoFisher Cat# P36930 DAPI ThermoFisher Cat# D1306 Mowiol 4-88 Sigma-Aldrich Cat# 475904 Propidium iodide solution Sigma-Aldrich Cat# P4864 PureLink RNase A ThermoFisher Cat# 12091021 Critical commercial assays Click-iT EdU Alexa Fluor 647 Cell Proliferation Kit for Imaging ThermoFisher Cat# C10340 Click-iT Nascent RNA Capture Kit ThermoFisher Cat# C10365 Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit ThermoFisher Cat# C10424 TruSeq ChIP Library Preparation Kit Illumina Cat# IP-202-1012 TruSeq Stranded Total RNA with RiboZero Gold Illumina Cat# RS-122-2301 Deposited data DRIP-seq data (HeLa cells) Hamperl et al., 2017 GEO: GSM2452072 qDRIP-seq data (HeLa cells) Crossley et al., 2020 GEO: GSE134084 OK-seq data (RFD in HeLa cells) Petryk et al., 2016 https://github.com/CL-CHEN-Lab/OK- Seq/tree/master/published_results/HeLa EdU-seq BRCA2-deficient H1299 Dagg et al., 2021 GEO: GSM4650337 MiDAS-seq data This paper GEO: GSE196163 EU-seq data This paper GEO: GSE196163 Experimental models: Cell lines H1299 +shBRCA2DOX cells (male origin) Zimmer et al., 2016 N/A HeLa cells (female origin) ATCC Cat# CCL-2 HeLa +RNaseH1-FLAGDOX (female origin) Sollier et al., 2014 N/A U2OS CyclinE (tet OFF) RAD52+/+ (female origin) Sotiriou et al., 2016 N/A U2OS CyclinE (tet OFF) RAD52-/- (female origin) Sotiriou et al., 2016 N/A U2OS T-REx GFP-RnaseH1D210N (female origin) Teloni et al., 2019 N/A Oligonucleotides siGENOME BRCA2 siRNA (SMARTpool) Horizon Discovery Cat# M-003462-01 ON-TARGETplus RAD52 siRNA (SMARTpool) Horizon Discovery Cat# L-011760-00-0005 BRCA2 siRNA Qiagen Cat# SI02653434 AllStar negative control Qiagen Cat# 1027281 TP53 siRNA Dharmacon Cat# GEHCU-000146 ON-TARGETplus non-targeting control pool Horizon Discovery Cat# D-001810-10-05 Software and algorithms GraphPad Prism GraphPad https://www.graphpad.com/ Olympus ScanR Image Analysis Software 3.0.0 Olympus https://www.olympus-lifescience.com/en/ microscopes/inverted/scanr/ TIBCO Spotfire TIBCO Software https://www.tibco.com/products/tibco- spotfire Fiji Schindelin et al., 2012 https://fiji.sc/ Bedtools Quinlan and Hall, 2010 https://bedtools.readthedocs.io/en/latest/ (Continued on next page) ll OPEN ACCESSArticle Molecular Cell 82, 3382–3397.e1–e7, September 15, 2022 e2

Techniques: Blocking Assay, Expressing, Two Tailed Test