Journal: Molecular cell

Article Title: Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

doi: 10.1016/j.molcel.2019.10.026

Figure Lengend Snippet: Figure 1. Replication Fork Stalling Induced by Co-transcriptional R-Loops Is Followed by Replication Restart via the SLX4-MUS81-RAD52- POLD3 Axis (A) Co-localization of PCNA and elongating RNA polymerase II (RNAPII pS2) in S phase nuclei of U2OS cells after 1 h of treatment with camptothecin (CPT; 100 nM) or pyridostatin (PDS; 10 mM), as determined by proximity ligation assay (PLA) and EdU-pulse labeling. Representative images (left panel) and quanti- fication of the percentage (right panel) of EdU+ and EdU cells with R5 PLA foci per nucleus are shown. EdU (10 mM) was added 10 min before CPT/PDS treatment. Where indicated, cordycepin (CORD; 50 mM) or DRB (100 mM) were added 2 h before CPT/PDS treatment. Data represent the means ± SDs, n = 3. Scale bar, 10 mm. (B) Effect of RNase H1 (RNH1) overexpression and transcription inhibition on replication fork slowing induced by CPT (100 nM) or PDS (10 mM) in U2OS T-REx/ RNH1-GFP cells. Top panel: experimental workflow of DNA fiber assays. Bottom panel: boxplot of values of the IdU:CldU tract length ratio obtained for indicated conditions (n R 200, whiskers: 10th–90th percentiles). RNH1 expression was induced with doxycycline (Dox). ns, not significant; ***p < 0.001; ****p < 0.0001 (Mann-Whitney test). (C) PDS and CPT induce sister fork asymmetry in a manner dependent on R-loop formation. Top panel: representative images of symmetric and asymmetric replication tracts of sister forks identified on DNA fibers in (B). Bottom panel: boxplot of the values of the sister fork IdU tract length ratio measured for the indicated conditions (n R 100, whiskers: 10th–90th percentiles). ns, not significant; ***p < 0.001; ****p < 0.0001 (Mann-Whitney test). (D) Effect of transcription inhibition and RNH1 overexpression on the frequency of reversed replication forks in U2OS T-REx/RNH1-GFP cells treated with CPT (100 nM) or PDS (10 mM) for 1 h. RNH1 expression was induced by the addition of Dox at 24 h before treatment. CORD (50 mM) was added 2 h before treatment. Data represent the means ± SDs, n = 3. p values: paired t test. (E) Western blot analysis of the extracts of U2OS cells transfected with indicated siRNAs. (F) Effect of the depletion of MUS81, EME1, SLX4, RAD52, and POLD3, respectively, on the rescue of CPT-induced replication fork slowing by PARP inhibition (PARPi) with 10 mM olaparib. Top panel: experimental workflow of DNA fiber assays. Bottom panel: boxplot of values of the IdU:CldU tract length ratio obtained for indicated conditions (n R 200, whiskers: 10th–90th percentiles). ns, not significant; ***p < 0.001; ****p < 0.0001 (Mann-Whitney test).

Article Snippet: The primary antibodies used for the immunofluorescence staining: MUS81 (MTA30 2G103) mouse monoclonal (sc-53382, Santa Cruz Biotechnology; 1:500 dilution), 53BP1 rabbit polyclonal (sc-22760, Santa Cruz Biotechnology; 1:500 dilution), Cyclin A (B-8) mouse monoclonal (sc-271682, Santa Cruz Biotechnology; 1:50 dilution), FANCD2 rabbit polyclonal (NB100-182, Novus Biologicals; 1:500 dilution).

Techniques: Proximity Ligation Assay, Labeling, Over Expression, Inhibition, Expressing, MANN-WHITNEY, Western Blot, Transfection

Journal: Molecular cell

Article Title: Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

doi: 10.1016/j.molcel.2019.10.026

Figure Lengend Snippet: Figure 3. Restart of R-Loop-Stalled Forks Requires Fork Cleavage by MUS81 Endonuclease (A) Genomic DNA breakage stimulated by PARPi in CPT- and PDS-treated U2OS T-REx/RNH1-GFP depends on DNA replication, transcription, and R-loop formation. Cells were treated with CPT (1 mM) or PDS (20 mM) for 5 h. Olaparib (PARPi; 10 mM), CORD (50 mM), and APH (5 mM), respectively, were added 2 h before CPT/PDS treatment. DNA breakage was monitored by pulsed-field gel electrophoresis (top panels) and quantified using ImageJ software (bottom panels). Data were normalized and represent the means ± SDs, n = 3. ****p < 0.0001 (unpaired t test). DSB, double-strand break. (B) Effect of the depletion of the indicated proteins on the level of DNA breakage in U2OS cells treated for 5 h with 1 mM CPT (black bars) or 20 mM PDS (gray bars) in the presence of PARPi. Genomic DNA was analyzed as in (A). Data represent the means ± SDs, n = 4. (C) The PARPi-mediated rescue of CPT- and PDS-induced replication fork slowing in U2OS cells requires MUS81 endonuclease activity. Top panel: experimental workflow of DNA fiber assays with U2OS cells stably transfected with WT MUS81 or MUS81(D338A/D339A) (MUS81Mut) cDNA constructs. Endogenous MUS81 was depleted with siRNA targeting MUS81 30 UTR (siMUS81UTR). Bottom panel: boxplot of the values of the IdU:CldU tract length ratio obtained for the indicated conditions (n R 200, whiskers: 10th–90th percentiles). ns, not significant; ****p < 0.0001 (Mann-Whitney test). (D) Western blot analysis of the extract of cells in (C). (E) Effect of CPT (100 nM) and PDS (10 mM) on replication fork progression in RA3331/E6E7/hTERT fibroblasts, complemented with either SLX4 WT or SLX4 DSAP cDNAs, before and after PARPi. Top panel: experimental workflow of DNA fiber assays. Bottom panel: boxplot of the values of the IdU:CldU tract length ratio obtained for the indicated conditions (n R 200, whiskers: 10th–90th percentiles). ns, not significant; ****p < 0.0001 (Mann-Whitney test). (F) Representative immunofluorescence images (top panel) and quantification (bottom panel) of MUS81 foci (green) colocalizing with FANCD2 foci (red) in U2OS cell nuclei (DAPI, blue) before and after treatment with CPT (100 nM) or PDS (10 mM) for 1 h. Data represent the means ± SDs, n = 3. Scale bar, 10 mm. (G) Effects of RNase H1 (RNH1) overexpression and transcription inhibition on the formation of MUS81 foci in U2OS T-REx/RNH1-GFP cells treated with CPT (100 nM) or PDS (10 mM) for 1 h. RNH1-GFP expression was induced 24 h before CPT/PDS treatment. CORD (50 mM) was added 2 h before the addition of CPT or PDS. Horizontal lines represent the means ± SEMs (n R 300). ns, not significant; ****p < 0.0001 (Mann-Whitney test). (H) Effects of SLX4 and EME1 depletions on the formation of FANCD2+ MUS81 foci in U2OS cells treated with CPT for 1 h. Data represent the means ± SDs, n = 3. See also Figure S4.

Article Snippet: The primary antibodies used for the immunofluorescence staining: MUS81 (MTA30 2G103) mouse monoclonal (sc-53382, Santa Cruz Biotechnology; 1:500 dilution), 53BP1 rabbit polyclonal (sc-22760, Santa Cruz Biotechnology; 1:500 dilution), Cyclin A (B-8) mouse monoclonal (sc-271682, Santa Cruz Biotechnology; 1:50 dilution), FANCD2 rabbit polyclonal (NB100-182, Novus Biologicals; 1:500 dilution).

Techniques: Nucleic Acid Electrophoresis, Software, Activity Assay, Stable Transfection, Transfection, Construct, MANN-WHITNEY, Western Blot, Over Expression, Inhibition, Expressing

Journal: Molecular cell

Article Title: Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

doi: 10.1016/j.molcel.2019.10.026

Figure Lengend Snippet: Figure 5. Restart of R-Loop-Stalled Forks Depends on the Catalytic Activity of the LIG4/XRCC4 Complex (A) Western blot analysis of the extracts of U2OS cells transfected with indicated siRNAs. (B) Effect of the depletion of the indicated proteins on replication fork progression in U2OS cells upon treatment with 100 nM CPT, and on the rescue of CPT-induced replication fork slowing by PARPi (10 mM olaparib). Top panel: experimental workflow of DNA fiber assays. Bottom panel: boxplot of the values of the IdU:CldU tract length ratio obtained for the indicated conditions (n R 200, whiskers: 10th–90th percentiles). ns, not significant; ****p < 0.0001 (Mann- Whitney test). (C) Effect of depletion of the indicated proteins on replication restart following the exposure of U2OS cells to CPT (100 nM) or PDS (10 mM). Top panel: experimental workflow of DNA fiber assays. Bottom panel: quantification of replication fork stalling events performed as in Figure 1G. Data represent the means ± SDs, n = 3. (D) LIG4/XRCC4 is required for mitotic DNA synthesis (MiDAS) in U2OS cells. MiDAS assay was performed as depicted in Figure S6B. The data points represent the number of EdU incorporation events per metaphase spread (n R 150). Horizontal lines represent the means ± SEMs. ns, not significant; ****p < 0.0001 (Mann- Whitney test). (E) Effect of CPT and PDS on replication fork progression in human fibroblasts expressing WT (LIG4WT, 1BR) or catalytically inactive (LIG4MUT, 411 BR) forms of LIG4, with or without PARPi. DNA fiber assays were performed as in Figure 3E. (F) The levels of spontaneous (DMSO) and CPT-induced DNA breakage in U2OS cells depleted for the indicated proteins. Cells were treated with 1 mM CPT for 5 h. Genomic DNA was analyzed as in Figure 3A. Data represent the means ± SDs, n = 4. (G) Effect of the depletion of LIG4 and RAD52 on the sensitivity of WT and MUS81 knockout (KO) HeLa Kyoto cells to PARPi, as determined by clonogenic assay. Data represent the means ± SDs, n = 3. Also see Figure S6.

Article Snippet: The primary antibodies used for the immunofluorescence staining: MUS81 (MTA30 2G103) mouse monoclonal (sc-53382, Santa Cruz Biotechnology; 1:500 dilution), 53BP1 rabbit polyclonal (sc-22760, Santa Cruz Biotechnology; 1:500 dilution), Cyclin A (B-8) mouse monoclonal (sc-271682, Santa Cruz Biotechnology; 1:50 dilution), FANCD2 rabbit polyclonal (NB100-182, Novus Biologicals; 1:500 dilution).

Techniques: Activity Assay, Western Blot, Transfection, MANN-WHITNEY, DNA Synthesis, Expressing, Knock-Out, Clonogenic Assay

Journal: Molecular cell

Article Title: Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

doi: 10.1016/j.molcel.2019.10.026

Figure Lengend Snippet: Figure 6. Restart of R-Loop-Stalled Forks Requires Reactivation of Transcription (A) Effect of DRB (100 mM) and CORD (50 mM) on replication restart following the treatment of U2OS cells with 100 nM CPT or 10 mM PDS. Top panel: experimental workflow of DNA fiber assays. Bottom panel: quantification of replication fork stalling events performed as in Figure 1G. Data represent the means ± SDs, n = 3. (B) Depletion of MUS81, LIG4, or ELL impairs the resumption of transcription following the exposure of U2OS cells to CPT. Nascent RNA strand production was quantified using 5-ethynyl uridine (EU; 1 mM) labeling during a 30-min treatment of cells with 100 nM CPT (or DMSO, control) and during a subsequent 30-min chase with CPT-free medium. The data represent the mean intensity of the EU signal in the nucleus. Horizontal lines represent median (n > 1,800). ****p < 0.0001 (Mann-Whitney test). (C) Effect of ELL depletion on replication restart following the treatment of U2OS cells with CPT (100 nM) or PDS (10 mM). Replication restart was quantified as in (A). (D) Effect of ELL depletion on replication fork progression in U2OS cells upon treatment with 100 nM CPT or 10 mM PDS, and on the rescue of CPT- or PDS- induced replication fork slowing by PARPi (10 mM olaparib). Top panel: experimental workflow of DNA fiber assay. Bottom panel: boxplot of the values of the IdU:CldU tract length obtained for the indicated conditions (n R 200, whiskers: 10th–90th percentiles). ns, not significant; ****p < 0.0001 (Mann- Whitney test). (E) Western blot analysis of the extracts of U2OS cells transfected with indicated siRNAs. (F) The levels of spontaneous (DMSO) and CPT-induced DNA breakage in U2OS cells depleted for the indicated proteins. Cells were treated with 1 mM CPT for 5 h. Genomic DNA was analyzed as in Figure 3A. Data represent the means ± SDs, n = 3. (G) Effect of ELL depletion on the sensitivity of WT and MUS81 KO HeLa Kyoto cells to PARPi as determined by clonogenic assay. Data represent the means ± SDs, n = 3. See also Figure S7.

Article Snippet: The primary antibodies used for the immunofluorescence staining: MUS81 (MTA30 2G103) mouse monoclonal (sc-53382, Santa Cruz Biotechnology; 1:500 dilution), 53BP1 rabbit polyclonal (sc-22760, Santa Cruz Biotechnology; 1:500 dilution), Cyclin A (B-8) mouse monoclonal (sc-271682, Santa Cruz Biotechnology; 1:50 dilution), FANCD2 rabbit polyclonal (NB100-182, Novus Biologicals; 1:500 dilution).

Techniques: Labeling, Control, MANN-WHITNEY, Western Blot, Transfection, Clonogenic Assay

Journal: Molecular cell

Article Title: Fork Cleavage-Religation Cycle and Active Transcription Mediate Replication Restart after Fork Stalling at Co-transcriptional R-Loops.

doi: 10.1016/j.molcel.2019.10.026

Figure Lengend Snippet: Figure 7. Model for the Resolution of R-Loop-Mediated TRCs The blockage of replication fork progression by an oncoming transcription complex results from the buildup of positive supercoiling within the intervening DNA region and the formation of an R-loop. Replication fork stalling leads to the assembly of RAD51 filament at the fork junction, which promotes fork reversal in conjunction with the DNA translocase ZRANB3. RECQ1 DNA helicase counteracts replication fork reversal to promote replication restart. In this pathway, RECQ5 DNA helicase disrupts the RAD51 filament on the stalled fork to facilitate fork cleavage by MUS81/EME1 endonuclease. This relieves the topological barrier in the DNA template, allowing ELL-mediated tran- scription restart. After re-annealing of the parental strands by RAD52 and sealing the nick in the parental duplex by the LIG4/XRCC4 complex, the re- activated transcription complex bypasses the replication-stalling site. This is followed by the POLD3-mediated restart of semiconservative DNA replication. It is assumed that after fork stalling, the replicative helicase CMG traverses the fork junction onto dsDNA via its ssDNA gate (not shown). After fork religation, CMG translocates back onto ssDNA to nucleate a functional replisome.

Article Snippet: The primary antibodies used for the immunofluorescence staining: MUS81 (MTA30 2G103) mouse monoclonal (sc-53382, Santa Cruz Biotechnology; 1:500 dilution), 53BP1 rabbit polyclonal (sc-22760, Santa Cruz Biotechnology; 1:500 dilution), Cyclin A (B-8) mouse monoclonal (sc-271682, Santa Cruz Biotechnology; 1:50 dilution), FANCD2 rabbit polyclonal (NB100-182, Novus Biologicals; 1:500 dilution).

Techniques: Functional Assay