MutSβ-MutLβ-FANCJ axis mediates the restart of DNA replication after fork stalling at cotranscriptional G4/R-loops

Abstract

Transcription-replication conflicts (TRCs) induce formation of cotranscriptional RNA:DNA hybrids (R-loops) stabilized by G-quadruplexes (G4s) on the displaced DNA strand, which can cause fork stalling. Although it is known that these stalled forks can resume DNA synthesis in a process initiated by MUS81 endonuclease, how TRC-associated G4/R-loops are removed to allow fork passage remains unclear. Here, we identify the mismatch repair protein MutSβ, an MLH1-PMS1 heterodimer termed MutLβ, and the G4-resolving helicase FANCJ as factors that are required for MUS81-initiated restart of DNA replication at TRC sites in human cells. This DNA repair process depends on the G4-binding activity of MutSβ, the helicase activity of FANCJ, and the binding of FANCJ to MLH1. Furthermore, we show that MutSβ, MutLβ, and MLH1-FANCJ interaction mediate FANCJ recruitment to G4s. These data suggest that MutSβ, MutLβ, and FANCJ act in conjunction to eliminate G4/R-loops at TRC sites, allowing replication restart.

MutSβ-MutLβ-FANCJ axis eliminates G4/R loops at sites of transcription-replication conflicts to promote replication restart.

INTRODUCTION

Genome replication is frequently challenged by various obstacles on the DNA template, which can halt replication fork progression. This phenomenon, known as DNA replication stress, can cause genomic instability, a hallmark of cancer (1). A potent source of DNA replication stress is active transcription if the transcription and replication machineries converge on the same DNA template (2). These transcription-replication conflicts (TRCs) are markedly increased upon overexpression of oncogenes such as CCNE1 and MYC that induce firing of DNA replication origins within highly transcribed genes in early S phase (3). Blockage of replication fork progression by head-on transcription results from the formation of an aberrant structure termed R-loop (4–6). In an R-loop, the nascent RNA transcript pairs with the template DNA strand behind the transcription complex, while the nontemplate strand loops out (7, 8). R- loops impede DNA replication by inducing a fork remodeling process known as replication fork reversal (6), which involves replisome disassembly and the pairing of nascent DNA strands to form a DNA duplex protected by BRCA2-stabilized RAD51 filament (9). R-loops tend to form in regions where the nontemplate strand is rich in guanines (Gs) with the potential to fold into G-quadruplex (G4) structures (10). These G4s may contribute to the formation of RNA:DNA hybrids during transcription, possibly by stabilizing the single-stranded DNA (ssDNA) tract within the resulting R-loop (8, 11). Intriguingly, these G4/R-loop structures, also termed G-loops, have been directly observed by electron microscopy following intracellular or in vitro transcription of plasmid regions containing a G-rich sequence in the nontemplate strand (12).

Reversed replication forks at sites of TRCs can resume DNA synthesis in a multistep process involving fork cleavage–religation cycles mediated by MUS81-EME1 endonuclease and the DNA ligase IV/XRCC4 complex coupled to transcription restart dependent on the RNA polymerase II (RNAPII) elongation factor ELL (6). A prerequisite for this sequential restart of transcription and replication at TRC sites is not only the removal of the R-loop but also the removal of G4 structures on the nontemplate strand, which, if persistent, would impede the progression of the reactivated replisome (13). While several human DNA helicases, including FANCJ, BLM, WRN, PIF1, and RTEL1, are known to unwind G4 structures in vitro (14), the specific helicase that removes G4 structures at sites of R-loop–mediated TRCs to allow replication fork passage is not known.

We have recently shown that the human mismatch repair (MMR) factor MutSβ, a heterodimer of MSH2 and MSH3 proteins, binds to G4 structures in vitro and regulates telomeric G4/R-loops in Alternative Lengthening of Telomeres (ALT) cancer cells to prevent telomere fragility and excision into extrachromosomal C-circles (15). The lack of MutSβ caused accumulation of RNA:DNA hybrids and G4s also at nontelomeric sites, suggesting a genome-wide role for this protein in G4/R-loop removal (15). In post-replicative MMR, MutSβ is responsible for the recognition of insertion/deletion loops generated by polymerase slippage, whereas base-base mismatches generated by nucleotide misincorporation are recognized by MutSα, a heterodimer of MSH2 and MSH6 proteins (16, 17). After mismatch recognition by MutSα or MutSβ, an MLH1-PMS2 heterodimer, known as MutLα, is recruited to trigger downstream steps of the canonical MMR pathway (18). MutLα acts as a latent endonuclease that is activated upon binding to the MutSα/MutSβ mismatch complex, generating nicks in the discontinuous DNA strand in a manner dependent on the DNA-loaded form of proliferating cell nuclear antigen (PCNA) (19). These strand breaks serve as an entry site for EXO1, which removes the mismatch in a 5′-to-3′ exonucleolytic reaction controlled by replication protein A (RPA), allowing nascent strand resynthesis (18, 19). In addition to MutLα, MLH1 forms two other heterodimers in mammalian cells: MutLβ (MLH1-PMS1) and MutLγ (MLH1-MLH3) (20, 21). While MutLγ is known to have an essential function in meiotic recombination and triplet repeat expansion (22–24), the function of MutLβ remains elusive. MutLβ (PMS1) lacks the motif required for endonuclease activity (19), suggesting a role other than that in mismatch correction.

Here, we provide several lines of evidence, suggesting that the restart of replication forks stalled by cotranscriptional G4/R-loops requires a coordinated action of MutSβ, MutLβ, and FANCJ. Our data suggest a model wherein MutSβ and MutLβ mediate FANCJ recruitment to G4s within R-loops at TRC sites for G4 unwinding. The data also suggest that G4 unwinding is required for the removal of R-loops.

RESULTS

MutSβ deficiency induces R-loop–dependent replication stress

U2OS cells lacking MutSβ were shown to accumulate G4 structures and R-loops not only at telomeres but also at nontelomeric loci (15), suggesting a genome-wide role for MutSβ in the regulation of these aberrant structures. To explore this further, we made use of a U2OS T-REx cell line inducibly expressing a green fluorescent protein (GFP)–tagged mutant of ribonuclease (RNase) H1, RNH1(D210N)-GFP, that binds but does not cleave RNA:DNA hybrids and thus enables visualization of the sites of R-loop formation as fluorescent nuclear foci (25, 26). Cells were transfected with MSH3 small interfering RNA (siRNA) to selectively eliminate MutSβ, and the expression of RNH1(D210N)-GFP was induced by adding doxycycline 24 hours before the analysis of individual cells by fluorescent microscopy (fig. S1A). Pulse labeling of nascent DNA with 5-ethynyl-deoxyuridine (EdU) was also carried out to identify S phase cells. We observed that MSH3 depletion increased the number of RNH1(D210N)-GFP foci in EdU⁺ cells but not in EdU⁻ cells, suggesting that MutSβ regulates R-loops formed specifically during S phase (fig. S1, B and C). We also analyzed R-loop levels in cells depleted of MSH6, which eliminates the MSH2-MSH6 heterodimer MutSα (fig. S1A). We found that the lack of MutSα did not markedly increase RNH1(D210N)-GFP foci either in EdU⁺ or in EdU⁻ cells (fig. S1C).

R-loop formation in S phase is promoted by head-on TRCs (4). Using proximity ligation assay (PLA) followed by quantitative image-based cytometry analysis, we found that the depletion of MSH3 but not MSH6 increased colocalization between elongating form of RNAPII (RNAPIIS2P, where S2P stands for phosphorylation of serine-2 in the C-terminal repeat domain of RNAPII) and the replisome component PCNA predominantly in EdU⁺ nuclei of U2OS cells (Fig. 1, A to D, and fig. S1D). This was prevented if cells were pretreated with the RNAPII transcription initiation inhibitor triptolide (TRP) (Fig. 1, A to D). MSH3 depletion also increased S phase–specific colocalization of elongating RNAPII with FANCD2 (fig. S1E), which is recruited to stalled replication forks (27). Together, these data suggest that a lack of MutSβ causes persistent TRCs.

Fig. 1. MutSβ deficiency causes persistent TRCs.

(A) Workflow of cell treatments for PLA to measure colocalization of elongating RNA polymerase II (RNAPIIS2P) and PCNA in cell nuclei. U2OS cells transfected with appropriate siRNA were pulse-labeled with EdU (10 μM) for 15 min. Where indicated, TRP (1 μM) was added 2 hours before cell harvest. (B) Western blot analysis of extracts of U2OS cells transfected with indicated siRNAs. (C) Representative images of RNAPIIS2P/PCNA PLA foci in EdU-positive nuclei of U2OS cells transfected with indicated siRNAs and incubated without [dimethyl sulfoxide (DMSO)] or with TRP as in (A). Scale bar, 10 μm. (D) Quantification of PLA foci in the images represented in (C). At least 741 nuclei were analyzed for each condition. Representative plot from two independent experiments yielding similar results is shown. Images from PLA with PCNA or RNAPIIS2P antibody only (negative control, in green) were also analyzed. Red lines represent median values. ****P < 0.0001; ns, not significant (Kruskal-Wallis test followed by Dunn’s multiple comparisons test).

Having determined that MutSβ deficiency induces S phase–specific accumulation of R-loops, we sought to investigate whether this condition has an impact on the progression of DNA replication forks. To this end, replication tracts in cells transfected with MSH3, MSH6, and control siRNA (siLuc), respectively, were sequentially pulse-labeled with halogenated thymidine (T) analogs 5-chloro-2′-deoxyuridine (CldU) and 5-iodo-2′-deoxyuridine (IdU) for 30 min each, and the stretches of nascent DNA were then visualized on DNA fiber spreads by indirect immunofluorescence (Fig. 2A). Using this so-called DNA fiber assay, we found that depletion of MSH3 but not MSH6 impaired replication fork progression in U2OS cells as revealed by shorter replication tracts compared to mock-depleted cells (Fig. 2, A and B). We also analyzed the progression of sister replication forks, which, under unperturbed conditions or upon a global replication slowing, would progress with similar rates, resulting in a symmetric pattern of replication tracts. However, in case of fork stalling by a barrier encountered on only one side of the replication origin, an asymmetric pattern of sister replication tracts would be observed, a phenotype termed sister fork asymmetry (Fig. 2C). Intriguingly, we could observe a substantially higher asymmetry of sister replication tracts in MSH3-deficient cells as compared to mock-depleted cells or cells depleted of MSH6 (Fig. 2D). Similar results were obtained with two other cell lines, RPE-1 and HeLa Kyoto (fig. S1, F to I). These data suggest that MutSβ deficiency increases the frequency of replication fork stalling events. In line with this conclusion, we observed that MSH3 depletion markedly induced the formation of micronuclei in U2OS cells (Fig. 2, E and F), a chromosome instability phenotype resulting from chromosome segregation defects in mitosis caused by regions of under-replicated DNA (28, 29). On the other hand, MSH6 depletion increased micronucleation frequency only slightly compared to MSH3-depleted cells (Fig. 2F).

Fig. 2. MutSβ deficiency induces R-loop-dependent replication stress.

(A to F) MSH3 depletion induces replication fork stalling and micronucleation in U2OS cells. (A) Top: Workflow of DNA fiber assays. Bottom: Representative images of replication tracts on DNA fibers of cells transfected with indicated siRNAs. (B) Quantification of the lengths of replication tracts (CldU + IdU) in the images represented in (A) (n ≥ 311). (C) Representative images of symmetric and asymmetric replication tracts of sister forks observed on DNA fiber spreads in (A). (D) Plot of the values of IdU tract length ratio of sister forks (sister fork ratio) obtained for indicated conditions (n ≥ 120). (E) Top: Workflow of cytokinesis-block micronucleus assay. Bottom: Representative images of binucleated cells with or without micronucleus (red arrow). (F) Quantification of the percentage of micronucleus-positive binucleated cells for indicated conditions. Data are means ± SEM (n = 3). (G to K) RNase H1 overexpression rescues replication fork stalling and micronucleation in MSH3-depleted cells. (G) Western blot analysis of extracts of U2OS T-REx [RNH1-GFP] cells transfected with indicated siRNAs and treated with (+) or without (−) doxycycline (Dox; 1 ng/ml) to induce expression of GFP-tagged RNase H1 (RNH1-GFP). (H) Workflow of DNA fiber assays. (I) Quantification of the lengths of DNA replication tracts for indicated conditions (n ≥ 402). (J) Plot of the values of sister fork ratio obtained for indicated conditions (n ≥ 211). (K) Top: Workflow of cytokinesis-block micronucleus assay. Bottom: Quantification of the percentage of micronucleus-positive binucleated cells for indicated conditions. Data are means ± SEM (n = 3). Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used in (B), (D), (I), and (J). ****P < 0.0001. One-way ANOVA with Tukey’s multiple comparisons correction was used in (F) and (K). ***P < 0.001 and *P < 0.05. All DNA fiber experiments were performed three times. Red lines represent median values. Scale bars, 10 μm. ns, not significant.

To investigate whether R-loops contribute to replication stress observed in cells lacking MutSβ, we used a stable U2OS T-REx cell line with doxycycline-regulated expression of the wild-type form of GFP-tagged RNase H1 (6). We found that RNase H1 overexpression, which eliminates R-loops (6), prevented fork slowing and sister fork asymmetry as well as micronucleation in MSH3-depleted cells (Fig. 2). These results suggest that fork stalling and chromosomal instability induced by MutSβ deficiency are caused by R-loops.

MutSβ is required for replication restart at R-loop–stalled replication forks

Replication fork reversal induced by G4/R-loop structures is counteracted by poly(adenosine 5′-diphosphate–ribose) polymerase 1 (PARP1)–regulated replication restart via the MUS81-LIG4-ELL axis (6). To determine how MutSβ averts R-loop–mediated replication stress, we first tested whether replication fork slowing in MSH3-depleted cells can be rescued by PARP inhibition, which promotes replication restart at G4/R-loop–stalled forks (6, 30, 31). By DNA fiber assay, we found that PARP inhibition with olaparib failed to restore normal fork progression in MSH3-depleted U2OS cells (fig. S2, A and B). In contrast, olaparib almost completely prevented replication fork stalling in cells depleted of RTEL1 helicase (fig. S2, A and B), known to suppress the accumulation of G4/R-loops (32). These data suggest that MutSβ might mediate the elimination G4/R-loops at sites of head-on TRCs to facilitate replication restart via the MUS81-LIG4-ELL axis. To explore this hypothesis, we used DNA fiber assay to analyze the effect of MSH3 depletion on replication fork dynamics upon treatment of cells with the G4-stabilizing ligand pyridostatin (PDS) or the DNA topoisomerase I inhibitor camptothecin (CPT), which promote R-loop formation and hence induce G4/R-loop–mediated fork stalling (6, 11, 32). As previously reported (6, 30, 31), we observed that the treatment of mock-depleted U2OS cells with PDS or CPT during the second pulse labeling with IdU resulted in replication tract shortening (IdU:CldU ratio < 1), which was largely prevented by addition of olaparib 2 hours before DNA fiber labeling (Fig. 3, A and B). However, PARP inhibition could not rescue PDS- or CPT-induced replication fork slowing in MSH3-depleted cells (Fig. 3B and fig. S2, C and D), mirroring the scenario in cells with defects in the MUS81-LIG4-ELL axis involved in restarting R-loop–stalled forks (6). In contrast, the depletion of MSH6 or RTEL1 had minimal impact on the rescue of PDS/CPT-induced fork slowing by PARP inhibition (Fig. 3B and fig. S2D). These results were reproduced in noncancerous RPE-1 cells (fig. S2E). Moreover, MSH3 or MSH6 depletion did not alter IdU:CldU ratio upon PARP inhibition alone in U2OS cells (Fig. 3B). Thus, we conclude that MutSβ but not MutSα plays a major role in the restart of DNA replication at sites of R-loop–mediated TRCs, possibly by mediating G4/R-loop removal. Consistent with the proposal that MutSβ promotes replication restart via the MUS81-LIG4-ELL axis, we found that MSH3 depletion did not further exacerbate replication fork stalling in MUS81 knockout HeLa Kyoto cells (fig. S2F to H) (6).

Fig. 3. MutSβ is required for replication restart at R-loop–stalled replication forks.

(A) Top: Workflow of DNA fiber assays with U2OS cells. The PARP inhibitor olaparib [10 μM; PARP inhibition (PARPi)/Pi] was added 2 hours before replication tract labeling and was also present during the labeling. PDS (10 μM) or CPT (100 nM) were added together with IdU. Bottom: Representative images of DNA replication tracts of U2OS cells transfected with control siRNA (siLuc) and treated as indicated. Scale bar, 10 μm. (B) Effect of depletion of MSH3 or MSH6 on replication fork progression in U2OS cells treated as indicated. Scatter plot of the IdU/CldU tract length ratio is shown. At least 328 replication tracts were scored in three independent experiments for each condition. Red lines represent median values. ****P < 0.0001 (Kruskal-Wallis test followed by Dunn’s multiple comparisons test). ns, not significant.

MutSβ mediates replication restart at R-loop–stalled replication forks through its G4-binding activity

We have recently shown that MutSβ specifically binds to G4 DNA structures in vitro (15). To explore the possible link between the G4-binding activity of MutSβ and its function in processing G4/R-loops, we sought to generate a mutant of MutSβ that is defective in G4 binding. Previous studies have shown that Y245S/K246E substitutions in the mismatch-binding domain (MBD) of MSH3 abolish the interaction of MutSβ with insertion/deletion loops and hairpin loops in vitro (33, 34). We therefore investigated whether these mutations also impair the binding of MutSβ to G4 DNA structures. To this end, we used a 5′-biotinylated 66-nucleotide oligomer DNA oligonucleotide composed of a stretch of 45 dTs followed by the d(GGGT)₄ sequence, which adopts a thermally stable parallel G4 topology in the presence of K⁺ ions (35). By pull-down assay with streptavidin-coated magnetic beads, we compared the binding of wild-type and Y245S/K246E variants of MutSβ to this G4-forming oligonucleotide after G4 folding in a KCl-based buffer. The binding reactions were also supplemented with the ssDNA-binding protein RPA to prevent nonspecific binding of MutSβ to the stretch of dTs. We found that wild-type MutSβ bound effectively to the G4-forming oligonucleotide both in the absence and presence of bound RPA, suggesting a specific binding (Fig. 4A). In contrast, the Y245S/K246E variant of MutSβ exhibited markedly reduced binding to this DNA structure when compared to the wild-type protein (Fig. 4A). To substantiate these findings, G4-binding activities of the wild-type and mutant forms of MutSβ were compared by electrophoretic mobility shift assay (EMSA) using a cyanine 3 (Cy3)–conjugated oligonucleotide, CEB1, known to fold into a parallel G4 structure, as confirmed by circular dichroism measurements (fig. S3A). In a protein titration experiment, we found that the Y245S/K246E MutSβ variant showed notably lower binding to the CEB1 G4 structure compared to the wild-type protein (Fig. 4B). Together, these data suggest that MutSβ binds to G4 DNA via its MBD.

Fig. 4. MBD of MutSβ mediates G4 DNA binding.

(A) Binding of wild-type (WT) and Y245S/K246E mutant forms of MutSβ (MutSβ^WT and MutSβ^Y245S/K246E) to biotinylated G4 DNA substrate b66-G4 in the absence or presence of RPA. The G4 oligonucleotide (20 nM) was preincubated with RPA (40 nM) before addition of MutSβ (50 nM). After incubation for 45 min at 4°C, DNA substrate was captured with streptavidin beads and bound proteins were detected by Western blotting. (B) Binding of WT and Y245S/K246E forms of MutSβ to CEB1 G4 detected by EMSA. Top: 30 nM Cy3-conjugated CEB1 G4 was incubated with indicated concentrations of MutSβ variants at 37°C for 10 min. DNA-protein complexes were separated from the free oligonucleotide by agarose gel electrophoresis. Bottom: Quantification of gels represented in the top panel. Data are means ± SD (n = 4).

To determine whether the binding of MutSβ to G4 is required for the restart of R-loop–stalled replication forks, we established U2OS T-REx cell lines expressing either wild-type or Y245S/K246E mutant form of a GFP-MSH3 chimera from a doxycycline-regulated cytomegalovirus (CMV) promoter (Fig. 5A). Silent mutations were introduced into both chimeras to confer siRNA resistance. We found that ectopic expression of wild-type MSH3 but not the Y245S/K246E variant could rescue replication fork slowing and sister fork asymmetry in cells depleted of endogenous MSH3 (Fig. 5, B to D). Notably, these findings could be reproduced in different cell clones (fig. S3, B to D). In addition, ectopic expression of the Y245S/K246E variant of MSH3 did not suppress the formation of micronuclei induced by depletion of endogenous MSH3, as seen with ectopic expression of wild-type MSH3 (Fig. 5E). In cells expressing the Y245S/K246E variant of MSH3 instead of endogenous MSH3, replication fork slowing induced by PDS or CPT could not be rescued by PARP inhibition, in contrast to cells ectopically expressing wild-type MSH3 (Fig. 5F and fig. S3E). These data suggest that the G4-binding activity of MutSβ is required for the restart of R-loop–stalled replication forks via the MUS81-LIG4-ELL axis.

Fig. 5. MutSβ mediates replication restart at R-loop–stalled replication forks through its G4-binding activity.

(A to E) Y245S/K246E substitutions in MSH3 induce replication fork stalling and micronucleation. (A) Western blot analysis of expression of WT or Y245S/K246E variants of GFP-tagged MSH3 in U2OS T-REx cells. Cells were transfected with indicated siRNAs for 72 hours and treated with doxycycline (0.4 ng/ml) in the past 24 hours or left untreated to induce transgene expression. (B) Experimental workflow of DNA fiber assays with cells in (A). (C) Quantification of replication tract lengths (CldU + IdU) for indicated conditions from three independent experiments (n ≥ 325). Horizontal lines represent median values. (D) Plot of the values of IdU tract length ratio of sister forks (sister fork ratio) obtained for indicated conditions (n ≥ 133). Horizontal lines represent median values. (E) Quantification of the frequency of micronuclei for indicated conditions. Data are means ± SEM (n = 3). (F) Y245S/K246E substitutions in MSH3 abolish the rescue of PDS-induced fork slowing by PARPi. Top: Workflow of DNA fiber assays with cells in (A). PARPi treatment was carried out as in Fig. 3A. PDS (10 μM) was added together with IdU. Bottom: Scatter plot of the values of IdU/CldU tract length ratio obtained for indicated conditions in two independent experiments (n ≥ 251). Horizontal lines represent median values. Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used in (C), (D), and (F). ****P < 0.0001. One-way ANOVA with Tukey’s multiple comparisons correction was used in (E). ***P < 0.001, **P < 0.01, and *P < 0.05. ns, not significant.

Replication restart at R-loop–stalled forks depends on MutLβ

In canonical MMR, mismatch recognition by MutSα or MutSβ is followed by the recruitment of MutLα (MLH1-PMS2 heterodimer), which triggers downstream steps of the repair process (18). MutLβ (MLH1-PMS1 heterodimer) is not required for MMR (20); however, a recent study showed that PMS1 depletion increases RNA:DNA hybrid levels in HeLa cells (36). Given these findings, we sought to investigate the possible roles of MutLα and MutLβ as factors that cooperate with MutSβ to promote the reactivation of R-loop–stalled forks via the MUS81-LIG4-ELL axis. We initially tested the effect of siRNA-mediated depletion of PMS1 or PMS2 on replication fork progression in U2OS cells. By DNA fiber assay, we found that depletion of either protein impaired replication fork progression as revealed by shorter replication tracts compared to mock-depleted cells (fig. S4, A and B). Notably, we observed a marked asymmetry of sister replication tracts in PMS1-depleted cells but not in PMS2-depleted cells (fig. S4C), suggesting that the lack of MutLβ but not MutLα induces replication fork stalling. Overexpression of RNase H1 completely rescued replication fork slowing and sister fork asymmetry in PMS1-depleted cells, while it had no impact on the reduced rate of replication fork progression in PMS2-depleted cells (Fig. 6, A to C). In addition, we found that depletion of PMS1 but not PMS2 increased the frequency of micronucleation, which could be also prevented by RNase H1 overexpression (Fig. 6D and fig. S4D). Together, these results suggest that MutLβ deficiency induces R-loop–dependent replication stress, whereas MutLα deficiency globally reduces replication fork velocity presumably due to defective MMR. In line with this conclusion, we observed a higher frequency of S phase–specific RNH1(D210N)-GFP foci and increased colocalization between elongating RNAPII and PCNA in PMS1-deficient cells compared to mock-depleted cells or cells depleted of PMS2 (Fig. 6, E and F). Ultimately, we tested the effect of PMS1 and PMS2 depletion on the rescue of PDS- or CPT-induced replication fork slowing by PARP inhibition in U2OS cells. We found that in cells depleted of PMS1, PARP inhibition did not restore the normal rate of fork progression upon treatment with PDS or CPT, whereas it did in cells depleted of PMS2 (Fig. 6G). These results suggest that the restart of R-loop–stalled forks via the MUS81-LIG4-ELL axis requires MutLβ.

Fig. 6. Replication restart at R-loop–stalled forks depends on MutLβ.

(A to D) PMS1 depletion induces R-loop–dependent replication stress. (A) Top: Workflow of DNA fiber assays with U2OS T-REx [RNH1-GFP] cells. Where required, overexpression of RNH1-GFP was induced with doxycycline (1 ng/ml). Bottom: Western blot analysis of extracts of U2OS T-REx [RNH1-GFP] cells transfected with indicated siRNAs. (B) Quantification of the lengths of DNA replication tracts (CldU + IdU) for indicated conditions in three independent experiments (n ≥ 456). (C) Plot of the values of IdU tract length ratio of sister forks (sister fork ratio) obtained for indicated conditions (n ≥ 92). (D) RNH1-GFP overexpression suppresses micronucleation induced by PMS1 depletion in U2OS T-REx [RNH1-GFP] cells. Data are means ± SEM (n = 3). (E) PMS1 depletion induces formation of nuclear foci of catalytically inactive form of RNase H1 [RNH1(D210N)-GFP]. Percentage of EdU⁺ cells with >10 RNH1(D210N)-GFP foci is plotted. Data are means ± SEM (n = 3). At least 300 cells per condition were analyzed. (F) PMS1 depletion induces colocalization of elongating RNA polymerase II (RNAPIIS2P) and PCNA in nuclei of U2OS cells as determined by PLA. Workflow of cell treatments was as in Fig. 1A. At least 178 nuclei were analyzed for each condition to quantify RNAPIIS2P/PCNA PLA foci. A representative plot from two independent experiments is shown. (G) PMS1 depletion abolishes the rescue of PDS/CPT-induced fork slowing by PARPi in U2OS cells. Left: Workflow of DNA fiber assays as in Fig. 3A. Right: Scatter plot of the values of IdU/CldU tract length ratio obtained for indicated conditions from two independent experiments (n ≥ 312). Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used in (B), (C), (F), and (G). ****P < 0.0001. One-way ANOVA with Tukey’s multiple comparisons correction was used in (D) and (E). ****P < 0.0001, ***P < 0.001, and *P < 0.05. Red lines represent median values. ns, not significant.

Replication restart at R-loop–stalled forks requires the helicase activity of FANCJ

Several DNA helicases have been shown to unwind G4 DNA structures in vitro (14). One of these helicases, FANCJ, has been identified as an interactor of MLH1 and PMS1 in human cells (37, 38). In addition, using an affinity pull-down assay with purified recombinant proteins, we found that FANCJ was bound to MutLβ (fig. S5). Therefore, we sought to investigate whether FANCJ is essential for the reactivation of R-loop–stalled forks via the MUS81-LIG4-ELL pathway. To this end, we first evaluated the effect of FANCJ deficiency on the progression of replication forks in unperturbed cells. We found that siRNA-mediated depletion of FANCJ reduced the rate of replication fork progression in U2OS cells, which was accompanied by a higher frequency of replication fork stalling events as revealed by sister fork asymmetry (fig. S6, A to C). A reduced replication speed and asymmetric progression of sister replication forks could be also observed in FANCJ knockout HeLa FlpIn T-REx (HeLa FIT) cells (fig. S6, D to G), generated by CRISPR-Cas9 technology (39). The replication fork slowing and sister fork asymmetry phenotypes induced by FANCJ depletion could be completely rescued by RNase H1 overexpression (Fig. 7, A to D), suggesting that, similar to MutSβ and MutLβ, FANCJ suppresses R-loop–mediated replication stress. Consistently, FANCJ-depleted cells exhibited an increased incidence of unresolved TRCs as reflected by a higher frequency of S phase–specific RNH1(D210N)-GFP foci and PLA foci between elongating RNAPII and PCNA compared to mock-depleted cells (Fig. 7, E and F). Moreover, we found that depletion of MSH3, PMS1, or MUS81 did not further exacerbate replication fork slowing and sister fork asymmetry in FANCJ knockout HeLa FIT cells, suggesting that all these proteins act in the same pathway for resolution of R-loop–stalled replication forks (fig. S6, H to J). Last, we tested the effect of FANCJ deficiency on the progression of replication forks in cells treated with PDS or CPT. We found that the unrestrained fork progression conferred by PARP inhibition in cells treated with these G4/R-loop–inducing drugs was abrogated by FANCJ deficiency (Fig. 7G and fig. S6, K and L). Together, these results suggest that FANCJ is needed for the restart of G4/R-loop–stalled forks via the MUS81-LIG4-ELL axis.

Fig. 7. Replication restart at R-loop–stalled forks depends on FANCJ.

(A to D) FANCJ depletion induces R-loop–dependent replication stress. (A) Workflow of DNA fiber assays with U2OS T-REx [RNH1-GFP] cells. Where required, overexpression of RNH1-GFP was induced with doxycycline (1 ng/ml) for 24 hours. (B) Western blot analysis of extracts of U2OS T-REx [RNH1-GFP] cells transfected with indicated siRNAs. (C) Quantification of the lengths of DNA replication tracts (CldU + IdU) for indicated conditions from three independent experiments (n ≥ 402). (D) Plot of the values of IdU tract length ratio of sister forks (sister fork ratio) obtained for indicated conditions (n ≥ 96). (E) FANCJ depletion induces accumulation of nuclear foci of catalytically inactive form of RNase H1 [RNH1(D210N)-GFP]. Cells were cultured, treated, and analyzed as depicted in fig. S1 (A to C). Percentage of EdU⁺ cells with >10 RNH1(D210N)-GFP foci is plotted. Data are means ± SEM (n = 3). At least 300 cells were analyzed in each experiment for each condition. (F) FANCJ depletion induces colocalization of elongating RNA polymerase II (RNAPIIS2P) and PCNA in the nuclei of U2OS cells lacking FANCJ as determined by PLA. At least 303 nuclei were analyzed for each condition to quantify RNAPIIS2P/PCNA foci. A representative plot of two independent experiments yielding similar results is shown. (G) FANCJ depletion abolishes the rescue of PDS/CPT-induced fork slowing by PARPi in U2OS cells. Top: Workflow of DNA fiber assays. Conditions for olaparib (PARPi) and PDS/CPT treatments were the same as in Fig. 3A. Bottom: Plot of the values of IdU/CldU tract length ratio obtained for indicated conditions in three independent experiments (n ≥ 291). Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used in (C), (D), (F), and (G). ****P < 0.0001. Paired t test was used in (E). **P < 0.01. Red lines represent median values. ns, not significant.

A K52R substitution in the ATP-binding pocket of FANCJ inactivates its ATPase/helicase function. This mutant was shown to be inefficient in G4 DNA unwinding both in vitro and in vivo (39, 40). To explore whether FANCJ exerts its function in counteracting G4/R-loop–dependent replication stress through its helicase activity, we stably transfected FANCJ knockout HeLa FIT cells with vectors expressing either wild-type FANCJ or its K52R mutant. We found that ectopic expression of wild-type FANCJ rescued the replication fork stalling phenotypes in both unchallenged and olaparib/PDS-treated FANCJ knockout cells, whereas the expression of the K52R mutant of FANCJ did not (Fig. 8, A to C). These data suggest that the helicase activity of FANCJ is required for its function in the restart of R-loop–stalled replication forks.

Fig. 8. Replication restart at R-loop–stalled forks requires the helicase activity of FANCJ.

(A) K52R substitution in helicase motif I of FANCJ induces sister fork asymmetry. Top: Workflow of DNA fiber assays with HeLa FlpIn T-REx (FANCJ^+/+) or CRISPR-Cas9–generated FANCJ knockout HeLa FIT cells (FANCJ^−/−). Where indicated, the expression of WT or K52R (helicase-dead) variants of FANCJ was induced with 1 μg/ml doxycycline (Dox) for 24 hours in FANCJ^−/− cells. Bottom: Plot of the values of IdU tract length ratio of sister forks (sister fork ratio) for indicated conditions. At least 100 tracts were scored for each condition in two independent experiments. (B) K52R substitution in helicase motif I of FANCJ abolishes the rescue of PDS-induced fork slowing by PARPi. Top: Workflow of DNA fiber assays with cells as in (A). Conditions for olaparib (PARPi) and PDS treatments were the same as in Fig. 3A. Bottom: Plot of the values of IdU/CldU tract length ratio obtained for indicated conditions. At least 300 replication tracts were measured for each condition in two independent experiments. (C) Western blot analysis of extracts of cells in (A). Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used in (A) and (B). ****P < 0.0001. Red lines represent median values. ns, not significant.

Reactivation of R-loop–stalled replication forks requires MLH1-FANCJ interaction

The findings presented thus far suggest a model wherein MutSβ and MutLβ recruit FANCJ to unwind G4 structures within R-loops at TRC sites, which is needed for replication restart via the MUS81-LIG4-ELL axis. To monitor the recruitment of FANCJ to the sites of G4s in cells, we sought to perform PLA using antibodies against FANCJ and G4 structures (BG4). Cells were pulsed with EdU to mark the S phase population and then subjected to PLA. We observed S phase–specific PLA foci in nuclei of U2OS cells under unperturbed conditions (Fig. 9, A and B, and fig. S7A). This phenotype was suppressed by a 2-hour pretreatment of cells with TRP, suggesting a dependence on transcription (Fig. 9B and fig. S7A). We then tested whether these PLA foci, indicating FANCJ/G4 colocalization, depend on MutSβ and MutLβ. We found that siRNA-mediated depletion of MSH3 and PMS1, respectively, lowered the number of PLA foci in U2OS cells without affecting FANCJ protein levels (Fig. 9, C and D). These results suggest that both MutSβ and MutLβ are required for the recruitment of FANCJ to G4s in vivo.

Fig. 9. Reactivation of R-loop–stalled replication forks requires MLH1-FANCJ interaction.

(A to D) Depletion of MSH3 or PMS1 impairs FANCJ recruitment to G4s in U2OS cells. (A) Representative images of FANCJ/BG4 PLA foci in EdU-positive nuclei. Where indicated, cells were treated with TRP (1 μM) for 2 hours. EdU was present the last 15 min. Scale bar, 10 μm. (B) Quantification of FANCJ/BG4 PLA foci in images represented in (A). (C) Quantification of FANCJ/BG4 PLA foci in U2OS cells lacking MSH3 or PMS1. A representative plot from two independent experiments is shown (n ≥ 1364). Images from PLA with BG4 or FANCJ antibody only were also analyzed. (D) Western blot analysis of extracts of cells in (C). (E and F) K141/142A substitutions in FANCJ abolish its recruitment to G4s in unchallenged cells. (E) Western blot analysis of extracts of FANCJ^+/+ or FANCJ^−/− HeLa FIT cells. Where indicated, the expression of WT or K141/142A variants of FANCJ was induced with doxycycline (1 μg/ml) in FANCJ^−/− cells. (F) Quantification of FANCJ/BG4 PLA foci in cells in (E). A representative plot from two independent experiments is shown (n ≥ 420). (G) K141/142A substitutions in FANCJ induce replication fork stalling in unchallenged cells. Top: Experimental workflow of DNA fiber assays with cells in (E). Bottom: Plot of the values of sister fork ratio obtained for indicated conditions in two independent experiments (n ≥ 202). (H) K141/142A substitutions in FANCJ abolish the rescue of PDS-induced fork slowing by PARPi. Top: Workflow of DNA fiber assays with cells in (E). Bottom: Plot of the values of IdU/CldU tract length ratio obtained for indicated conditions in two independent experiments (n ≥ 189). Kruskal-Wallis test followed by Dunn’s multiple comparisons test was used in (B), (C), and (F) to (H). ****P < 0.0001. Red lines represent median values. ns, not significant.

Previous studies identified a FANCJ variant containing K141/142A substitutions that render FANCJ defective in its interaction with MLH1 and abolish FANCJ recruitment to sites of DNA crosslinks without affecting its helicase activity (38, 41, 42). Intriguingly, we found that FANCJ knockout HeLa FIT cells expressing the K141/142A mutant of FANCJ had a defect in the recruitment of FANCJ to G4s, as revealed by PLA (Fig. 9, E and F). This observation supports the notion that the physical interaction between FANCJ and MLH1 is required for efficient recruitment of FANCJ to G4 sites. In addition, complementation with the K141/142A mutant of FANCJ failed to rescue replication fork slowing and sister fork asymmetry in FANCJ knockout cells (Fig. 9G and fig. S7B). Moreover, in these cells, PARP inhibition did not restore the normal rate of replication fork progression upon PDS treatment, as it did in FANCJ knockout cells complemented with wild-type FANCJ (Fig. 9H). These data suggest that MLH1-FANCJ interaction is required for replication restart at G4/R-loop–stalled replication forks.

DISCUSSION

Cotranscriptional R-loops, a major source of DNA replication stress, are formed preferentially in actively transcribed regions where the nontranscribed strand contains runs of guanines that can fold into G4 structures. Specific G4 ligands that stabilize G4s increase R-loop levels in these G-rich regions and induce R-loop–dependent stalling of DNA replication forks (6, 11, 32). This suggests that the formation of G4s in the displaced nontranscribed strand stabilizes R-loops and promotes their extension. Our earlier studies have shown that replication forks stalled by G4/R-loops can resume DNA synthesis by a process involving fork cleavage-religation cycles mediated by MUS81-EME1 endonuclease and the LIG4-XRCC4 complex, which presumably relieve the torsional stress in the DNA template generated by transcription-replication encounters, allowing for transcription and replication restart (6). Here, we identify three additional factors that act in this DNA repair process: the G4-resolving helicase FANCJ; the MutSβ heterodimer (MSH2-MSH3), which specifically binds to G4s; and the MutLβ heterodimer (MLH1-PMS1), which physically interacts with FANCJ. We show that replication restart at G4/R-loops depends on the helicase activity of FANCJ, the G4-binding activity of MutSβ, and the binding of FANCJ to MLH1. Moreover, we present evidence suggesting that MutSβ and MutLβ mediate the recruitment of FANCJ to the sites of G4s in cell nuclei. On the basis of these findings, we propose a model wherein MutSβ, MutLβ, and FANCJ act in conjunction to eliminate G4 structures within R-loops at TRC sites, thereby facilitating replication restart via the MUS81-LIG4-ELL axis (Fig. 10). It is possible that the unwinding of G4s is essential for the helicase-mediated removal of R-loops. In line with this assumption, we found that depletion of MSH3, PMS1, and FANCJ, respectively, induced R-loop accumulation in S phase cells (Figs. 6E and 7E and fig. S1C). Notably, we have recently identified the DEAD-box helicase DDX17 as a factor that might be involved in R-loop unwinding at sites of R-loop–mediated TRCs to promote replication restart via the MUS81-LIG4-ELL axis (43). Consistently, DDX17 has been found to efficiently unwind synthetic R-loop structures in vitro (43). To initiate strand separation, DEAD-box helicases are loaded directly onto the duplex region of an RNA, aided by a proximal single-stranded nucleic acid region that does not have to be covalently linked to the helix (44). Thus, in the context of an R-loop structure, DDX17 loading on the RNA:DNA hybrid could be mediated through its binding to the displaced nontranscribed strand (Fig. 10). In this scenario, the presence of G4s in the single-stranded region of the R-loop could potentially block DDX17 loading, thereby preventing R-loop unwinding (Fig. 10).

Fig. 10. Model for the elimination of G4/R-loops at TRC sites and the consequences of its failure.

MutSβ binds to G4s in the non-transcribed strand through its MBD domain and recruits MutLβ-FANCJ complex for G4 unwinding. This facilitates the loading of the DDX17 helicase on the ssDNA loop and the subsequent unwinding of the R-loop, leading to the sequential restart of transcription and replication via the MUS81-LIG4-ELL pathway. Failure of G4 unwinding impairs DDX17 loading, leading to persistent fork stalling. Sβ, MutSβ; Lβ, MutLβ; FJ, FANCJ; CMG, CDC45-MCM2-7-GINS helicase.

The K141/K142A substitutions in FANCJ were also shown to abolish the binding of FANCJ to G4 in vitro (45). However, the K141/K142A mutant of FANCJ displayed G4-unwiding activity if the DNA substrate contained 5′-ssDNA overhang for FANCJ loading (45). Moreover, we have found that the recruitment of FANCJ to G4 sites is mediated by MutSβ and MutLβ. Thus, it is unlikely that the observed defect of FANCJ K141/K142A-expressing cells in restarting G4/R-loop–stalled forks is primarily caused by the inability of the FANCJ mutant to recognize G4.

Our data implicating FANCJ in G4 unwinding during the restart of R-loop–stalled forks are consistent with the previous observation of a FANCJ requirement for unrestrained DNA synthesis in HLTF-deficient cells exposed to hydroxyurea (HU) (46), since HU-induced replication fork stalling is known to be caused by R-loops (47). Moreover, abrogation of HU-induced fork reversal by HLTF depletion has been shown to trigger replication restart via the MUS81-LIG4-ELL axis (47). However, a recent study using high-resolution microscopy has shown that FANCJ, in an interplay with RPA, regulates spontaneously formed G4s on newly unwound DNA at replication forks, immediately behind the MCM helicase and before nascent DNA synthesis (48). The increased frequency of these G4/replisome structures in FANCJ-depleted cells was associated with a reduction in EdU incorporation both at nuclear and individual replisome level, particularly upon PDS treatment (48). However, it is not clear to what extent these G4s contribute to replication fork stalling observed in FANCJ-deficient cells by DNA fiber assay, as this phenotype was largely rescued by overexpression of RNase H1, suggesting a dependence on G4/R-loop structures (Fig. 7D).

Cells defective in MLH1-FANCJ interaction or FANCJ helicase activity show hypersensitivity to DNA-crosslinking agents such as mitomycin C (MMC) (38). However, the underlying molecular mechanism is not clear. In addition to DNA interstrand crosslinks, MMC was also found to induce accumulation of R-loop–dependent DNA damage in human cells (49). Moreover, replication fork stalling induced by MMC can be rescued by inhibition of fork reversal (50), a phenomenon observed for R-loop–stalled forks (6). Thus, it will be interesting to explore whether MMC sensitivity of FANCJ-deficient cells stems from a defect in restarting R-loop–stalled forks via the MUS81-LIG4-ELL axis.

MATERIALS AND METHODS

Plasmid constructions

The E. coli strain DH10B was used for all plasmid constructions (Thermo Fisher Scientific). The vectors for inducible expression of wild-type and mutant (Y245S/K246E) variants of human MSH3 were constructed using the plasmid pEGFP-c1-hMSH3 that expresses MSH3 as an N-terminal fusion with GFP (34). The Afe I/Kpn I fragment of pEGFP-c1-hMSH3 including the GFP-MSH3 chimera was introduced into the multiple cloning site (Pme I/Kpn I) of the plasmid pAIO to generate a regulatable transcription unit with a CMV enhancer/promoter followed by two tet operators (51). Using the QuickChange II Site-Directed Mutagenesis Kit (Agilent Technologies), a set of silent mutations was introduced into the MSH3 open reading frame (ORF) to confer resistance to siMSH3#1 in the resulting transcript. Ultimately, this pAIO-GFP-MSH3 construct was subjected to another round of site-directed mutagenesis to introduce Y245S/K246E mutations into the MSH3 gene (pAIO-GFP-MSH3-Y245S/K246E). Using site-directed mutagenesis, K141/142A variant of FANCJ was generated in the FANCJ-pDONR221 vector (39). To construct the transfer vector for generation of a bacmid encoding for His-tagged PMS1, the PMS1 ORF was amplified by polymerase chain reaction (PCR) using the primers 5′-TATATGGATCCATGCATCATCATCATCACCACGGTTCTGGTATGAAACAATTGCCTGCGG-3′ and 5′-ATATATAAGCTTTCATGTAGTTTCTGGAAGATAGGT-3′, which introduce Bam HI and Hind III sites, respectively, and the forward primer also introduces an N-terminal 6xHis-tag to PMS1. As a template for PCR, the plasmid pFastBac1-hPMS1 was used (20). The resulting PCR product was cut with Bam HI and Hind III and cloned in pFastBac1 (pFastBac1-6xHis-hPMS1).

Generation of stable cell lines

U2OS T-REx cells were transfected with pAIO-GFP-MSH3 or pAIO-GFP-MSH3-Y245S/K246E constructs using TransIT-X2 reagent (Mirus). Transfected cells were selected in the presence of puromycin (1 μg/ml; InvivoGen). Clones were isolated after 10 to 14 days of growth in the selection medium. The expression of the desired proteins upon doxycycline induction was tested by Western blotting with anti-GFP and anti-MSH3 antibodies. CRISPR-Cas9–generated FANCJ HeLa FlpIn T-REx (FANCJ^−/− HeLa FIT) knockout cells (39) were cotransfected with pDONR221-based vector expressing either wild-type, K52R, or K141/142A variant of FANCJ, as well as an Flp-In-compatible expression vector plasmid (pOG44) encoding for Flp recombinase, using TransIT-X2 reagent (Mirus). Transfected FANCJ^−/− HeLa FIT cells were selected in the presence of blasticidin (15 μg/ml; InvivoGen) and hygromycin (150 μg/ml; InvivoGen). The expression of these constructs allowing complementation of FANCJ-deficient cells with FANCJ variants upon doxycycline induction was tested by Western blotting with anti-FANCJ antibody.

Cell culture

U2OS (HTB-96), HeLa Kyoto (CVCL_1922), and RPE-1 cells (CRL-4000) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific) supplemented with 5% fetal calf serum (FCS; Thermo Fisher Scientific) and streptomycin/penicillin (100 U/ml) at 37°C in a humidified incubator containing 5% CO₂. U2OS T-REx–derived cell lines were grown in DMEM supplemented with Tet-free approved 5% FCS, streptomycin/penicillin (100 U/ml), puromycin (1 μg/ml), and hygromycin B (50 μg/ml; InvivoGen) at 37°C in a humidified incubator containing 5% CO₂. U2OS T-REx cell lines carrying RNH1-GFP and RNH1(D210N)-GFP transgenes, respectively, were described previously (25). Expression of RNase H1 variants was induced by addition of doxycycline (catalog no. 631311, Takara Bio) to a concentration of 1 ng/ml for 24 hours. Ectopic expression of MSH3 variants was tuned to be comparable with the level of endogenous MSH3 by titration of doxycycline concentration. Typically, doxycycline was added to a concentration of 0.4 ng/ml for 24 hours. HeLa FIT (52) and FANCJ^−/− HeLa FIT cell lines were grown in DMEM supplemented with 5% FCS at 37°C in a humidified incubator at a 5% CO₂-containing atmosphere. Stably transfected FANCJ^−/− HeLa FIT cells were cultured in DMEM supplemented with 5% FCS, blasticidin (15 μg/ml; InvivoGen), and hygromycin (150 μg/ml; InvivoGen). Expression of constructs in FANCJ^−/− HeLa FIT was induced by doxycycline (1 μg/ml) for 24 hours. The following drugs were used at the indicated final concentrations, unless stated otherwise: PDS pentahydrochloride (10 μM; catalog no. 4763, Tocris Biosciences), CPT (100 nM; catalog no. C9911, Sigma-Aldrich), Olaparib (10 μM; catalog no. S1060, Selleckchem), cytochalasin B (2 μg/ml; catalog no. C6762, Sigma-Aldrich), and TRP (1 μM; catalog no. T3652, Sigma-Aldrich).

siRNA transfections

Transfections of siRNAs (a final concentration of 40 nM) were done at 30 to 40% confluency using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. Twenty-four hours after siRNA transfection, the medium was exchanged with fresh medium. The sequences of the sense strand of siRNA duplexes are listed in table S1. For ectopic expression of MSH3 variants in stable U2OS T-REx cell lines, endogenous MSH3 was depleted by transfection of siMSH3#1 for a total time of 72 hours. Forty-eight hours after siRNA transfection, doxycycline (0.4 ng/ml) was added to induce expression of MSH3 variants for 24 hours. In the figures where siMSH3 and siPMS1 are indicated, data for siMSH3#1 and siPMS1#1, respectively, are shown.

Preparation of cell extracts and Western blot analysis

Cells that were trypsinized and washed with 1× phosphate-buffered saline (PBS) were resuspended in lysis buffer [50 mM tris-HCl buffer (pH 7.5), 120 mM NaCl, 20 mM NaF, 1 mM EDTA, 6 mM EGTA, 15 mM Na-pyrophosphate, and 0.5% (v/v) NP-40] supplemented with protease inhibitor cocktail (cOmplete, EDTA free; Sigma-Aldrich) and phosphatase inhibitor cocktail (PhosSTOP, Roche). After sonication for 5 min with a diagenode sonicator, cell lysate was centrifuged at 11,000 rpm for 30 min at 4°C to separate cellular debris from the soluble cell fraction. Bradford assay was carried out for the determination of protein concentration. A total of 30 to 50 μg of total protein from whole-cell extracts were loaded onto 8 to 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels. Proteins separated by SDS-PAGE were blotted onto a Hybond-P polyvinylidene difluoride membrane (GE Healthcare) in a semi-dry transfer apparatus at 56 mA for 2 hours. After Ponceau staining, the membranes were blocked in 2% ECL Prime blocking reagent (GE Healthcare) in TBS-T [20 mM tris-HCl (pH 7.4), 150 mM NaCl, and 0.1% (v/v) Tween 20] for 30 min at room temperature (RT). Then, the membranes were incubated in primary antibodies diluted in 2% ECL blocking solution at 4°C overnight. After three washes in TBS-T, the membranes were incubated in the corresponding horseradish peroxidase (HRP)–coupled secondary antibody for 1 hour at RT. Then, the membranes were washed three times with TBS-T, and protein bands were detected using ECL Western blotting substrate (Pierce). The following primary antibodies were used for immunoblotting: GFP rabbit polyclonal (1:2000; ab290, Abcam), MSH3 (H300) rabbit polyclonal (1:1000; sc-11441, Santa Cruz Biotechnology), MSH6 (clone 44/MSH6) mouse monoclonal (1:1000; 610918, BD Transduction Laboratories), PMS1 (E-3) mouse monoclonal (1:1000; sc-515302, Santa Cruz Biotechnology), PMS2 (clone A16-4) mouse monoclonal (1:1000; 556415, BD Pharmingen), BRIP1/FANCJ rabbit polyclonal (1:1000; NBP1-31883, Novus Biologicals), TFIIH p89 (S-19) rabbit polyclonal (1:1000; sc-293, Santa Cruz Biotechnology), glyceraldehyde-3-phosphate dehydrogenase (0411) mouse monoclonal (1:1000; sc-47724, Santa Cruz Biotechnology), β tubulin (clone TUB 2.1) mouse monoclonal (1:1000; T-4026, Sigma-Aldrich), and γ-tubulin rabbit polyclonal (1:1000; T-3559, Sigma-Aldrich). The following secondary antibodies used for Western blotting are as follows: goat anti-rabbit IgG-HRP (1:5000; A5050, Sigma-Aldrich) and goat anti-mouse IgG HRP (1:10,000; A4416, Sigma-Aldrich).

Detection of R-loops by monitoring RNH1(D210N)-GFP foci

U2OS T-REx [RNH1(D210N)-GFP] cells were grown on autoclaved glass coverslips. RNH1(D210N)-GFP expression was induced by addition of doxycycline to a concentration of 1 ng/ml for 24 hours. To mark the S phase population, cells were pulse-labeled with EdU (10 μM) for 15 min. After a brief wash with ice-cold 1× PBS, cells were pre-extracted using ice-cold pre-extraction solution [25 mM Hepes-NaOH (pH 7.7), 50 mM NaCl, 1 mM EDTA, 3 mM MgCl₂, 300 mM sucrose, and 0.5% (v/v) Triton X-100] on ice for 10 min and then briefly washed with pre-extraction solution that does not contain Triton X-100. The cells were fixed with 4% (v/v) formaldehyde for 15 min at RT in the dark, and after several washes with 1× PBS, cells were permeabilized with 0.5% (v/v) Triton X-100/1× PBS for 5 min at RT. Following three washes with 1× PBS, cells were blocked in 3% bovine serum albumin (BSA)/1× PBS for 30 min at RT. To visualize EdU incorporation, Click-iT Plus EdU Alexa Fluor 594 Imaging reaction was performed according to the manufacturer’s protocol (Thermo Fisher Scientific). After washing with 3% BSA/1× PBS and then 1× PBS, the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/ml) in distilled water and mounted using Fluoromount-G (Invitrogen). All images were captured with a fluorescent microscope at ×63 magnification (Leica microscope, model DM6B coupled to the DMC 2900 digital camera). At least 300 nuclei were analyzed using ImageJ. DAPI signal was used for the identification of the nuclei, and EdU signal was used for the determination of S phase cells. The percentage of EdU-positive cells with more than 10 RNH1(D210N)-GFP foci was calculated.

In situ PLA

Cells were grown on autoclaved glass coverslips. To mark the S phase population, cells were pulse-labeled with EdU (10 μM) for 15 min. Cells were briefly washed with ice-cold 1× PBS and incubated with ice-cold 0.5% (v/v) Triton X-100/1× PBS supplemented with protease inhibitor cocktail (cOmplete, EDTA free; Sigma-Aldrich) for 10 min on ice for pre-extraction. After washing with 1× PBS three times, cells were fixed with 4% (v/v) formaldehyde for 15 min at RT in the dark. Following three washes with 1× PBS, coverslips were incubated with prechilled 100% (v/v) methanol for 20 min at −20°C. Following three washing steps with 1× PBS, cells were permeabilized with 0.2% (v/v) Triton X-100/1× PBS for 10 min at RT. Cells were washed with 1× PBS and incubated with blocking solution (5% BSA/1× PBS) for 30 min at RT. Primary antibody (diluted in blocking solution) incubation was performed overnight at 4°C. After several washes of coverslips with 1× PBS, PLA was carried out according to the manufacturer’s protocol (Sigma-Aldrich). Briefly, coverslips were incubated with anti-Mouse MINUS and anti-Rabbit PLUS PLA probes (Sigma-Aldrich) for 1 hour at 37°C. Coverslips were then washed twice in Wash Buffer A [0.01 M tris (pH 7.4), 0.15 M NaCl, and 0.05% (v/v) Tween 20] for 5 min by gentle shaking. The ligation step where minus and plus probes were ligated was performed for 30 min at 37°C. After two washes in wash buffer A for 5 min by gentle shaking, incubation with “Duolink In Situ Detection Reagents Green” (Sigma-Aldrich) or “Duolink In Situ Detection Reagents Red” (Sigma-Aldrich) was carried out for the amplification step for 100 min. Then, coverslips were washed twice with 1× wash buffer B [0.2 M tris-HCl (pH 7.5) and 0.1 M NaCl] for 10 min and once with 0.01× wash buffer B for 5 min by gentle shaking at RT. After a brief incubation with blocking solution, the Click-iT EdU reaction was performed according to the manufacturer’s protocol for the visualization of EdU incorporation (Thermo Fisher Scientific). After washing with blocking buffer and then 1× PBS, the cells were counterstained with DAPI (1 μg/ml) in distilled water and mounted using Fluoromount-G (Invitrogen). The representative images were acquired with a Leica DM6B fluorescent microscope at ×63 magnification. For determination of the number of PLA foci, images were captured automatically on an IX83 microscope (Olympus) equipped with ScanR imaging platform using 40×/0.9 numerical aperture objective. Number of PLA foci per nucleus from at least 1000 nuclei marked by DAPI signal was determined using ScanR analysis software. EdU signal was used for the determination of S phase cells. The primary antibodies used in PLA are: PCNA rabbit polyclonal (1:500; catalog no. ab18197, Abcam), FANCD2 rabbit polyclonal (1:500; NB100-182, Novus Biologicals), RNAPII (H5) mouse monoclonal (1:1000; 920204, BioLegend), BRIP1/FANCJ rabbit polyclonal (1:500; NBP1-31883, Novus Biologicals), DNA/RNA G-quadruplex [BG4] recombinant mouse monoclonal (1:1000; Ab00174-1.1, Absolute Antibody).

DNA fiber assay

For measurements of replication fork dynamics under different conditions, well-established DNA fiber spreading assay was performed as previously described (6). Briefly, cells were sequentially pulse-labeled with two thymidine analogs: 30 μM CldU (Sigma-Aldrich) for 30 min and, after three washes with 1× PBS, 250 μM IdU (Sigma-Aldrich) for 30 min. After labeling, cells were washed three times with ice-cold 1× PBS to cease nucleotide incorporation and trypsinized. Cells were then resuspended in 1× PBS to a concentration of 2.5 × 10⁵ cells/ml. The labeled cells were diluted with unlabeled cells in 1:1 ratio, and 2.5 μl of cell suspension was spotted on the glass slide and air-dried for 1 min. The cells were then lysed on the glass slide with 7.5 μl of lysis buffer [200 mM tris-HCl (pH 7.5), 50 mM EDTA, and 0.5% (w/v) SDS]. The slides were then incubated in horizontal position for exactly 9 min at RT without moving and then tilted at 15° to 45° to allow the DNA fibers to slowly stretch along the slide in 1 to 3 min. The slides were air-dried at RT and fixed with methanol/acetic acid (3:1) at 4°C overnight. DNA fibers on glass slides were denatured with 2.5 M HCl for 1 hour at RT and then washed four times with 1× PBS and blocked with blocking solution [2% BSA/1× PBS/0.1% (v/v) Tween 20] for 10 min at RT. Subsequently, DNA fibers were incubated with primary antibodies diluted in blocking solution for 2.5 hours at RT. Rat monoclonal anti–5-bromo-2′-deoxyuridine (BrdU) antibody (1:500; ab6326, Abcam) was used to detect CldU and mouse monoclonal anti-BrdU antibody (1:100; 347580, BD Biosciences) was used to detect IdU. Following five washes with PBS-T [1× PBS/ 0.2% (v/v) Tween 20], slides were incubated for 1 hour in the dark at RT with the secondary antibodies diluted in the blocking solution. Donkey anti-rat Cy3 (1:150; 712-166-153, Jackson ImmunoResearch) was used against anti-CldU antibody and goat anti-mouse Alexa 488 (1:300; A110334, Thermo Fisher Scientific) was used against anti-IdU antibody. The secondary antibody incubation was followed by five washes with PBS-T, and then the slides were air-dried before the mounting of coverslips with Antifade Gold (Invitrogen) mounting medium. Images of labeled replication tracts were acquired with a Leica DM6B fluorescent microscope (63×/1.40 oil immersion), and their lengths were measured by using segmented line tool of ImageJ.

Cytokinesis-block micronucleus assay

Cells were cultured on autoclaved coverslips and the culture medium was supplemented with cytochalasin B (2 μg/ml; micro-filament assembly inhibitor; C6762, Sigma Aldrich) for 16 hours before the cell harvest to block cytokinesis. The cells were fixed with 4% (v/v) formaldehyde for 15 min at RT in the dark, and after several washes with 1× PBS, cells were counterstained with DAPI (1 μg/ml) in distilled water and mounted using Fluoromount-G (Invitrogen). Images were captured with a Leica DM6B fluorescent microscope at ×63 magnification, and the percentage of binucleated cells with micronuclei was determined. At least 250 binucleated cells were analyzed in each experiment.

Protein expression and purification

RPA was produced in bacteria and purified as previously described (53). MutSβ, MutLβ, and FANCJ proteins were produced in Spodoptera frugiperda (Sf9) insect cells using the Bac-to-Bac Baculovirus Expression System (catalog no. 10359016, Thermo Fisher Scientific) as previously described (20, 34, 39). Purification of MutSβ (wild-type and Y245S/K246E mutant) carrying a hexahistidine tag on MSH3 was described previously (34). For purification of MutLβ, a 400-ml culture of Sf9 cells at 1 × 10⁶ cells/ml was infected with freshly amplified recombinant baculoviruses encoding for hMLH1 and His-hPMS1, respectively. Seventy-two hours after infection, cells were harvested by centrifugation at 480g for 20 min at 4°C. The cell pellet was resuspended in 50 ml of lysis buffer A [25 mM Hepes-NaOH (pH 7.6), 300 mM NaCl, 10% (v/v) glycerol, and 2 mM 2-Mercaptoethanol] supplemented with 10 mM imidazole, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 1× protease inhibitor cocktail (Roche cOmplete, EDTA free). Cells were disrupted by sonication on ice (four pulses of 30 s, 60% power, 70% cycle), and the lysate was centrifuged for 15 min at 3200g, 4°C, followed by ultracentrifugation at 100,000g for 1 hour. The supernatant was loaded onto a 5-ml HisTrap FF column (GE HealthCare) equilibrated with buffer A/10 mM imidazole. After binding, the column was washed with 75 ml of buffer A/20 mM imidazole followed by 75 ml of buffer A/40 mM imidazole. MutLβ protein was eluted with buffer A/250 mM imidazole. Peak fractions with MutLβ, determined by SDS-PAGE, were pooled, diluted 1.2× with buffer B [25 mM Hepes-NaOH (pH 7.6), 0.1 mM EDTA, 10% (v/v) glycerol, and 1 mM dithiothreitol (DTT)] to a final concentration of NaCl of 250 mM. The sample was loaded onto a 5-ml HiTrap Heparin column (GE HealthCare) equilibrated with buffer B/250 mM NaCl. The column was washed with 100 ml of buffer B/250 mM NaCl. Bound proteins were eluted with a 50-ml linear gradient of NaCl from 250 to 800 mM. Peak fractions were pooled, diluted 3× with buffer B, and loaded onto a 1-ml Mono Q 5/50 GL column (GE HealthCare) equilibrated with buffer B/150 mM NaCl. The column was washed with 15 ml of buffer B/150 mM NaCl. Bound proteins were eluted with a 20-ml linear gradient of 150 to 550 mM NaCl in buffer B. Peak fractions were pooled; dialyzed against 25 mM Hepes-NaOH (pH 7.5), 20% (v/v) glycerol, 0.1 mM EDTA 110 mM NaCl, and 0.1 mM PMSF; aliquoted; and stored at −80°C.

FANCJ was affinity purified via its N-terminal Flag-tag using anti-FLAG M2 Magnetic beads (Sigma-Aldrich). Approximately 5 × 10⁸ cells were infected with freshly amplified recombinant baculoviruses encoding for N-terminally Flag-tagged FANCJ. Forty-eight hours after infection, the cells were harvested by centrifugation at 480g for 20 min at 4°C. The pellet was resuspended in 4× pellet volume (cca. 20 ml) of lysis/binding buffer C [10 mM tris-HCl (pH 7.4), 130 mM NaCl, 1.0% (v/v) Triton X-100, 10 mM NaF, 10 mM NaH₂PO₄, 10 mM Na₄P₂O₇, and 10% (v/v) glycerol] supplemented with 0.1 mM PMSF and 1× protease inhibitor cocktail (Roche cOmplete, EDTA free). Cells were lysed for 60 min at 4°C on a rotary shaker. The lysate was centrifuged at 4°C for 15 min at 3200g, followed by 45 min at 20,000g. The supernatant was mixed with 640 ml of slurry anti-Flag M2 magnetic beads equilibrated with buffer C followed by incubation for 2 hours at 4°C on a rotary shaker. After binding, the beads were separated from the supernatant with unbound proteins by using a magnetic rack. The resin was washed three times with 30 ml of buffer D [50 mM tris-HCl (pH 7.4), 0.5% (v/v) NP-40, and 10% (v/v) glycerol] supplemented with 500 mM NaCl and 0.1 mM PMSF, followed by one wash in buffer D supplemented with 150 mM NaCl and 0.1 mM PMSF. After quantitative removal of all wash buffer, the beads were eluted three times, each with 1 ml of elution buffer E [25 mM tris-HCl (pH7.4), 100 mM NaCl, 0.1% (v/v/) Tween 20, 10% (v/v) glycerol, 0.1 mM PMSF, 2 mM 2-mercaptoethanol, and 3xFlag-peptide (150 μg/ml)]. Concentration of purified proteins was determined by densitometric analysis of SDS-PAGE gels calibrated by BSA.

His pull-down assay

Purified MutLβ protein carrying a 6xHis tag on the PMS1 subunit (1 μg) was incubated with purified Flag-tagged FANCJ (0.5 μg) in a buffer (500 μl) containing 50 mM tris-HCl (pH 7.5), 0.1% (v/v) NP-40, 10% (v/v) glycerol, 150 mM NaCl, 1 mM 2-mercaptoethanol, and 10 mM imidazole for 2 hours at 4°C. Subsequently, Ni-NTA beads (20 μl; QIAGEN) were added and incubation was continued for 2 hours at 4°C. Beads were washed three times with the above buffer. The bound proteins were eluted with 200 mM imidazole (50 μl) and analyzed by Western blotting using anti-MLH1 and anti-FANCJ antibodies.

Biotin pull-down assay

To form a G4 structure substrate, 5′-biotin–labeled b66-G4 oligonucleotide (5′-Biotin-TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGGGTGGGTGGGTGGGTTTTTT-3′) was diluted to a concentration of 1 μM in NEBuffer 4 (1×) and then heat-denatured at 95°C for 5 min followed by 20-min incubation at 85°C and slow cooling to RT. Binding reactions (50 μl) were performed in a buffer containing 50 mM tris-HCl (pH 7.5), 120 mM KCl, and 0.1% (v/v) Triton X-100. Initially, b66-G4 at a concentration of 20 nM was incubated with streptavidin-coupled M280 Dynabeads (10 μl; Life Technologies) for 15 min at RT. Where indicated, RPA (40 nM) was added to the mixture and incubation was carried out in a rotary shaker for 15 min at 4°C to coat the single-stranded part of b66-G4. Mixtures were then supplemented with 50 nM wild-type or Y245S/K246E mutant MutSβ, and incubation was continued for 45 min at 4°C. Last, the beads were washed three times with the binding buffer, and bound proteins were analyzed by Western blotting.

Electrophoretic mobility shift assay

Cy3-labeled CEB1 G4 oligonucleotide (5′-Cy3-AGGGGGGAGGGAGGGTGG-3′) was folded into G4 structure in 100 mM KCl by heating at 95°C for 5 min, followed by slow cooling to RT. The folded oligonucleotide was incubated at a concentration of 30 nM with increasing concentrations of MutSβ (wild type or Y245S/K246E mutant) in 20 mM tris-HCl (pH 7.5), 5 mM MgCl₂, 1 mM DTT, 75 mM NaCl, and BSA (0.05 mg/ml) at 37°C for 10 min. The assembled complexes were separated from free oligonucleotide on 0.8% agarose gel in 1× TBE (Tris-Borate-EDTA) buffer supplemented with 10 mM KCl at 70 V for 35 min at 4°C. Gels were scanned on an Amersham Typhoon Biomolecular Imager scanner (Cytiva) and quantified with Multi Gauge version 3.2 (Fujifilm).

Circular dichroism spectroscopy

Circular dichroism spectra of 7.5 μM CEB1 G4 oligonucleotide in 10.5 mM KCl were collected from 200 to 330 nm with a spectral bandwidth of 1 nm using a Chirascan CD Spectrometer. The spectrum shown is an average of three technical replicates.

Statistical analysis

Statistical analysis was performed with GraphPad Prism 9 software using either paired Student’s t test, repeated-measures one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons correction, two-tailed nonparametric Kruskal-Wallis test followed by Dunn’s multiple comparisons test, or two-tailed nonparametric Mann-Whitney test, where appropriate. Details of statistics in each experiment can be found in the corresponding figure legends.

Supplementary Materials

This PDF file includes:

Figs. S1 to S7

Table S1