Transcription-replication Conflicts Research Articles

RNA biogenesis and RNA metabolism factors as R-loop suppressors: a hidden role in genome integrity.

Genome integrity relies on the accuracy of DNA metabolism, but as appreciated for more than four decades, transcription enhances mutation and recombination frequencies. More recent research provided evidence for a previously unforeseen link between RNA and DNA metabolism, which is often related to the accumulation of DNA-RNA hybrids and R-loops. In addition to physiological roles, R-loops interfere with DNA replication and repair, providing a molecular scenario for the origin of genome instability. Here, we review current knowledge on the multiple RNA factors that prevent or resolve R-loops and consequent transcription-replication conflicts and thus act as modulators of genome dynamics.

RNA biogenesis and RNA metabolism factors as R-loop suppressors: a hidden role in genome integrity.

Genome integrity relies on the accuracy of DNA metabolism, but as appreciated for more than four decades, transcription enhances mutation and recombination frequencies. More recent research provided evidence for a previously unforeseen link between RNA and DNA metabolism, which is often related to the accumulation of DNA-RNA hybrids and R-loops. In addition to physiological roles, R-loops interfere with DNA replication and repair, providing a molecular scenario for the origin of genome instability. Here, we review current knowledge on the multiple RNA factors that prevent or resolve R-loops and consequent transcription-replication conflicts and thus act as modulators of genome dynamics.

Immune evasion: An imperative and consequence of MYC deregulation.

MYC has been implicated in the pathogenesis of a wide range of human tumors and has been described for many years as a transcription factor that regulates genes with pleiotropic functions to promote tumorigenic growth. However, despite extensive efforts to identify specific target genes of MYC that alone could be responsible for promoting tumorigenesis, the field is yet to reach a consensus whether this is the crucial function of MYC. Recent work shifts the view on MYC's function from being a gene-specific transcription factor to an essential stress resilience factor. In highly proliferating cells, MYC preserves cell integrity by promoting DNA repair at core promoters, protecting stalled replication forks, and/or preventing transcription-replication conflicts. Furthermore, an increasing body of evidence demonstrates that MYC not only promotes tumorigenesis by driving cell-autonomous growth, but also enables tumors to evade the host's immune system. In this review, we summarize our current understanding of how MYC impairs antitumor immunity and why this function is evolutionarily hard-wired to the biology of the MYC protein family. We show why the cell-autonomous and immune evasive functions of MYC are mutually dependent and discuss ways to target MYC proteins in cancer therapy.

Genome-wide Mapping of Topoisomerase Binding Sites Suggests Topoisomerase 3α (TOP3A) as a Reader of Transcription-Replication Conflicts (TRC).

Both transcription and replication can take place simultaneously on the same DNA template, potentially leading to transcription-replication conflicts (TRCs) and topological problems. Here we asked which topoisomerase(s) is/are the best candidate(s) for sensing TRC. Genome-wide topoisomerase binding sites were mapped in parallel for all the nuclear topoisomerases (TOP1, TOP2A, TOP2B, TOP3A and TOP3B). To increase the signal to noise ratio (SNR), we used ectopic expression of those topoisomerases in H293 cells followed by a modified CUT&Tag method. Although each topoisomerase showed distinct binding patterns, all topoisomerase binding signals positively correlated with gene transcription. TOP3A binding signals were suppressed by DNA replication inhibition. This was also observed but to a lesser extent for TOP2A and TOP2B. Hence, we propose the involvement of TOP3A in sensing both head-on TRCs (HO-TRCs) and co-directional TRCs (CD-TRCs). In which case, the TOP3A signals appear concentrated within the promoters and first 20 kb regions of the 5' -end of genes, suggesting the prevalence of TRCs and the recruitment of TOP3A in the 5'-regions of transcribed and replicated genes.

Dormant origin firing promotes head-on transcription-replication conflicts at transcription termination sites in response to BRCA2 deficiency

BRCA2 is a tumor suppressor protein responsible for safeguarding the cellular genome from replication stress and genotoxicity, but the specific mechanism(s) by which this is achieved to prevent early oncogenesis remains unclear. Here, we provide evidence that BRCA2 acts as a critical suppressor of head-on transcription-replication conflicts (HO-TRCs). Using Okazaki-fragment sequencing (Ok-seq) and computational analysis, we identified origins (dormant origins) that are activated near the transcription termination sites (TTS) of highly expressed, long genes in response to replication stress. Dormant origins are a source for HO-TRCs, and drug treatments that inhibit dormant origin firing led to a reduction in HO-TRCs, R-loop formation, and DNA damage. Using super-resolution microscopy, we showed that HO-TRC events track with elongating RNA polymerase II, but not with transcription initiation. Importantly, RNase H2 is recruited to sites of HO-TRCs in a BRCA2-dependent manner to help alleviate toxic R-loops associated with HO-TRCs. Collectively, our results provide a mechanistic basis for how BRCA2 shields against genomic instability by preventing HO-TRCs through both direct and indirect means occurring at predetermined genomic sites based on the pre-cancer transcriptome.

Human DNA topoisomerase I poisoning causes R loop-mediated genome instability attenuated by transcription factor IIS.

DNA topoisomerase I can contribute to cancer genome instability. During catalytic activity, topoisomerase I forms a transient intermediate, topoisomerase I-DNA cleavage complex (Top1cc) to allow strand rotation and duplex relaxation, which can lead to elevated levels of DNA-RNA hybrids and micronuclei. To comprehend the underlying mechanisms, we have integrated genomic data of Top1cc-triggered hybrids and DNA double-strand breaks (DSBs) shortly after Top1cc induction, revealing that Top1ccs increase hybrid levels with different mechanisms. DSBs are at highly transcribed genes in early replicating initiation zones and overlap with hybrids downstream of accumulated RNA polymerase II (RNAPII) at gene 5'-ends. A transcription factor IIS mutant impairing transcription elongation further increased RNAPII accumulation likely due to backtracking. Moreover, Top1ccs can trigger micronuclei when occurring during late G1 or early/mid S, but not during late S. As micronuclei and transcription-replication conflicts are attenuated by transcription factor IIS, our results support a role of RNAPII arrest in Top1cc-induced transcription-replication conflicts leading to DSBs and micronuclei.

A PARP1-TIMELESS alliance in cancer therapy

In a recent paper in Nature, Petropoulos etal.1 report that PARP1 acts together with the replisome components TIMELESS and TIPIN to protect the genome from transcription-replication conflicts, which has important implications for the clinical use of PARP inhibitors.

Chromosome organization shapes replisome dynamics in Caulobacter crescentus

DNA replication in bacteria takes place on highly compacted chromosomes, where segregation, transcription, and repair must occur simultaneously. Within this dynamic environment, colocalization of sister replisomes has been observed in many bacterial species, driving the hypothesis that a physical linker may tether them together. However, replisome splitting has also been reported in many of the same species, leaving the principles behind replisome organization a long-standing puzzle. Here, by tracking the replisome β-clamp subunit in live Caulobacter crescentus, we find that rapid DNA segregation can give rise to a second focus which resembles a replisome, but does not replicate DNA. Sister replisomes can remain colocalized, or split apart to travel along DNA separately upon disruption of chromosome inter-arm alignment. Furthermore, chromosome arm-specific replication-transcription conflicts differentially modify replication speed on the two arms, facilitate the decoupling of the two replisomes. With these observations, we conclude that the dynamic chromosome organization flexibly shapes the organization of sister replisomes, and we outline principles which can help to reconcile previously conflicting models of replisome architecture.

Replication-Transcription Conflicts: A Perpetual War on the Chromosome.

DNA replication and transcription occur in all living cells across all domains of life. Both essential processes occur simultaneously on the same template, leading to conflicts between the macromolecular machines that perform these functions. Numerous studies over the past few decades demonstrate that this is an inevitable problem in both prokaryotic and eukaryotic cells. We have learned that conflicts lead to replication fork reversal, breaks in the DNA, R-loop formation, topological stress, and mutagenesis, and they can ultimately impact evolution. Recent studies have also provided insight into the various mechanisms that mitigate, resolve, and allow tolerance of conflicts and how conflicts result in divergent pathological consequences across divergent species. In this review, we summarize current knowledge regarding the outcomes of encounters between replication and transcription machineries and explore how these clashes are dealt with across species.

Abstract 5784: Structure-based drug design against caPCNA for selective cancer chemotherapy development

Abstract Proliferating cell nuclear antigen (PCNA) is critical to DNA replication and repair processes, and it is also a proliferation biomarker in a variety of human tumors. A unique cancer-associated isoform of the protein, caPCNA, has been previously identified, allowing for rational-based drug discovery studies to develop leads with the potential for selective therapeutic targeting of cancer cells. Several strategies have been employed to develop agents targeting caPCNA, including peptide and small molecule-based inhibitors, but the success in developing therapeutically tractable compounds has been limited so far. Here, we present the crystal structures and corresponding biochemical characterizations of our novel small molecule-based caPCNA inhibitors, which bind into the PIP-box binding pocket of PCNA. One of our lead compounds is now an investigational new drug that we observed selectively kills cancer cells, and it appears to induce replication stress, apoptosis and increase cancer cell sensitivity to genotoxic agents, while these effects are not observed in non-malignant cell controls. This caPCNA inhibitor is orally administrable, metabolic stable and it suppresses tumor growth as a monotherapy or as a combination treatment, and it has entered clinical trials in the United States. PCNA plays a critical role in temporarily dislodging RNA polymerase II from transcription replication conflict sites, to enable the replication fork to proceed. Thus, structure-based drug discovery approaches to target transcription replication conflict resolution machinery, such as PCNA protein, may open a novel therapeutic avenue for exploiting this cancer-selective vulnerability. Citation Format: Jennifer Jossart, Pouya Haratipour, Long Gu, Carone M. li, Robert Lingeman, Yilun Liu, Robert J. Hickey, Linda H. Malkas, Jeff J. Perry. Structure-based drug design against caPCNA for selective cancer chemotherapy development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5784.

Abstract 6583: Developing a selective cancer chemotherapy by small molecule-based targeting of PCNA protein

Abstract Proliferating cell nuclear antigen (PCNA) is a proliferation biomarker in a variety of human tumors and has critical cellular functions in DNA replication and repair. We previously identified a unique cancer-associated isoform of the protein, termed caPCNA, which allows for structure-based drug discovery studies to develop inhibitors for selective therapeutic targeting of cancer cells. Distinct strategies have been used to develop caPCNA targeting agents, which includes peptide and small molecule-based compounds, although the success in developing therapeutically tractable compounds has been limited so far. Here, we present the crystal structures and corresponding biochemical characterizations of our novel small molecule-based caPCNA inhibitors, which bind into the PIP-box binding pocket of PCNA. Our lead compound selectively kills cancer cells, and it appears to induce replication stress, apoptosis and increase cancer cell sensitivity to genotoxic agents, while these effects are not observed in non-malignant cell controls. This caPCNA inhibitor is orally administrable, metabolic stable and it suppresses tumor growth as a monotherapy or as a combination treatment and is now an investigational new drug in Phase 1 clinical trials. PCNA plays a critical role enabling the replication fork to proceed from transcription replication conflict sites, by temporarily dislodging RNA polymerase II. Thus, structure-based drug discovery against caPCNA may open a novel therapeutic avenue for the development of selective chemotherapeutics, through targeting a cancer cell’s vulnerability to unresolved transcription replication conflicts. Citation Format: Jennifer Jossart, Long Gu, Pouya Haratipour, Caroline Li, Robert Lingeman, Robert Hickey, Linda Malkas, John J. Perry. Developing a selective cancer chemotherapy by small molecule-based targeting of PCNA protein [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6583.

Abstract 3367: R-loop type determines the orientation of transcription-replication conflicts attenuated by transcription factor IIS

Abstract During human Topoisomerase I (Top1) catalytic activity, a transient Top1-DNA cleavage complex (Top1cc) forms to allow DNA relaxation. Top1ccs can be stabilized by antitumor Top1 poisons, such as clinically-effective camptothecin analogs. Camptothecin-stabilized Top1ccs can lead to R-loop-dependent transcription-replication conflicts (TRCs), single-end DNA double-strand breaks (seDSBs) and genome instability in cancer cells. To define the genomic location of TRCs and the underlying mechanisms, we integrated genomic maps of Top1cc-induced DNA cleavage by END-seq and DNA:RNA hybrids by DRIP, determined the cell-cycle specificity of micronuclei formation and the effects of elongation transcription factor IIS (TFIIS). We have defined the genomic sites of both seDSBs and increased R-loop levels (gains) induced by Top1ccs. seDSBs are highly enriched at regions with R-loop gains at highly-transcribed genes in early replicating zones. Kinetics analyses of R-loops and genomic ChIP data of RNA polymerase II (RNAPII) revealed that R-loop gains can either be upstream or downstream of RNAPII. TFIIS effects showed that downstream R-loop gains can follow RNAPII backtracking and arrest. seDSB strandness showed that co-directional TRCs occur mainly at downstream hybrid-associated RNAPII, whereas head-on TRCs occur mainly at upstream hybrid-associated RNAPII. Therefore, Top1ccs can increase two structurally-different R-loops and R-loop-associated backtracked RNAPII causes co-directional TRCs at higher rate than head-on collisions. Moreover, Top1ccs trigger micronuclei formation at G1/early S, but not late S/G2, which are attenuated by TFIIS. Thus, our findings establish a role of RNAPII backtracking at highly-transcribed genes in Top1cc-induced co-directional TRCs and micronuclei formation. The findings can accelerate the development of new effective strategies to treat cancer patients. (Supported by Associazione Italiana per la Ricerca sul Cancro, Milan, Italy - IG 2019 - ID. 23032 to C.G. and by the European Union - NextGenerationEU through the Italian Ministry of University and Research under PNRR - M4C2-I1.3 Project PE_00000019 "HEAL ITALIA" to G.C., CUP J33C22002920006). Citation Format: Renée C. Duardo, Jessica Marinello, Marco Russo, Sara Morelli, Simona Pepe, Federico Guerra, Belen Gomez-González, Andrés Aguilera, Giovanni Capranico. R-loop type determines the orientation of transcription-replication conflicts attenuated by transcription factor IIS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3367.

Abstract 1875: Enhanced lung cancer treatment using AOH1996, a potent PCNA inhibitor

Abstract AOH1996 targets a cancer-associated form of PCNA (caPCNA) by altering the protein-protein interface between caPCNA and its many binding partners. AOH1996 does this by inserting into the PCNA-interacting protein-box (PIP-box) pocket that is, in part, defined by the interdomain connecting loop (IDCL), the site of interaction between PCNA and most of its many binding partners. As a result, AOH1996 inhibits PCNA functions such as DNA replication, DNA repair, and transcription-replication conflict (TRC) resolution leading to cell cycle arrest and apoptosis. These effects are cancer specific and AOH1996 has little effect on non-cancerous cells even at 6-fold the effective dose in cancer cells. AOH1996 was developed in our lab and is currently in Phase I human clinical trials. Six patients have been enrolled in the trial and have not experienced AOH1996-related toxicities as the doses have been escalated to levels found to be efficacious in preclinical animal studies. Resistance to EGFR tyrosine kinase inhibitors (TKIs) poses a significant challenge in the treatment of non-small cell lung cancer (NSCLC) patients harboring EGFR activating mutations. Initially, EGFR TKIs like gefitinib, erlotinib, afatinib and osimertinib deliver remarkable responses, inducing tumor regression and improving overall survival. However, the emergence of resistance mechanisms, such as secondary EGFR mutations and bypass signaling pathway activation, hampers the efficacy of these drugs over time. The functional relationship between PCNA and EGFR has implications for the treatment of lung cancer patients with activating EGFR mutations. Membrane-localized EGFR modulates the PCNA-centric process of DNA replication through signaling pathways such as the Ras-Raf-Mek-Erk, PI3K/AKT, and PLCγ1/PKC signaling pathways. Nucleus-localized EGFR enhances PCNA stability by phosphorylating PCNA on tyrosine 211 (Y211), which prevents its degradation. In addition, phosphorylation at Y211 by EGFR results in decreased association of PCNA with MutS, which results in reduced mismatch repair (MMR). In this study, we combine AOH1996 and osimertinib as an innovative approach to treating NSCLC cancers with activating EGFR mutations and find that the drug combination: 1.) enhances the killing of NSCLC cell lines with activating EGFR mutations and acquired resistance to TKIs; 2.) enhances the destabilization of PCNA on chromatin; and 3.) causes changes in the localization and colocalization of PCNA and EGFR. We conclude that the combination of AOH1996 and osimertinib holds much potential as an improved treatment regimen for lung cancer patients with activating EGFR mutations. Citation Format: Robert G. Lingeman, Robert J. Hickey, Linda H. Malkas. Enhanced lung cancer treatment using AOH1996, a potent PCNA inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1875.

Abstract 2112: Identification of biomarkers associated with sensitivity to a novel PCNA inhibitor (AOH1996) by CRISPRi screening

Abstract Proliferating cell nuclear antigen (PCNA) plays an essential role in regulating DNA synthesis and repair and is essential for cancer cell growth and survival. We discovered a cancer-associated isoform of PCNA (caPCNA), which is ubiquitously and highly expressed in a broad range of cancer cells and tumor tissues but is not significantly expressed in non-malignant cells. Further studies of this novel caPCNA isoform as an anti-cancer drug target led to the identification of a cancer distinct region of PCNA which is partly delineated by the L126-Y133 peptide sequence and the discovery of a small molecule (AOH1996), which binds to this cancer distinct region as shown by crystallization studies. By targeting caPCNA, AOH1996 selectively kills cancer cells by interfering with the resolution of transcription-replication conflicts (TRC). Importantly, it causes no discernable toxicity at 6 times of its effective dose in mice. AOH1996 is currently in a phase 1 clinical trial for adult solid tumors at City of Hope (IND 138273). To identify genes that may be used as biomarkers for patient selection or potential targets for combination therapies, we screened a CRISPR interference (CRISPRi) library, which targets genes that are involved in cell signaling and cell cycle regulation. After culturing transduced cells in the presence of DMSO or 250 nM AOH1996 (corresponding to IC20) in triplicates for 14 generations, we measured the relative abundance of each guide RNA (gRNA) construct. Significant changes in the abundance of the gRNA constructs in AOH1996-treated cells relative to that in DMSO-treated cells were determined by the MAGeCK method. The target genes of the depleted or enriched gRNAs are predominantly involved in DNA repair, cell cycle regulation, and transcription regulation, which is consistent with the action mechanism of AOH1996. We validated the effect of these genes on sensitivity to AOH1996 and identified a panel of target proteins whose up or down-regulation is associated with sensitivity to AOH1996. Some of these proteins are targets of known chemotherapeutic drugs, which may be used in combination with AOH1996 in the clinic. Citation Format: Long Gu, Ming li, Chun-Wei Chen, Robert J. Hickey, Linda H. Malkas. Identification of biomarkers associated with sensitivity to a novel PCNA inhibitor (AOH1996) by CRISPRi screening [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2112.

Transcription–replication conflicts underlie sensitivity to PARP inhibitors

An important advance in cancer therapy has been the development of poly(ADP-ribose) polymerase (PARP) inhibitors for the treatment of homologous recombination (HR)-deficient cancers1–6. PARP inhibitors trap PARPs on DNA. The trapped PARPs are thought to block replisome progression, leading to formation of DNA double-strand breaks that require HR for repair7. Here we show that PARP1 functions together with TIMELESS and TIPIN to protect the replisome in early S phase from transcription–replication conflicts. Furthermore, the synthetic lethality of PARP inhibitors with HR deficiency is due to an inability to repair DNA damage caused by transcription–replication conflicts, rather than by trapped PARPs. Along these lines, inhibiting transcription elongation in early S phase rendered HR-deficient cells resistant to PARP inhibitors and depleting PARP1 by small-interfering RNA was synthetic lethal with HR deficiency. Thus, inhibiting PARP1 enzymatic activity may suffice for treatment efficacy in HR-deficient settings.

WRNIP1 prevents transcription-associated genomic instability

R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified fork protection factors and DNA damage response proteins as regulators of R-loop dynamics. In this study, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity.

WRNIP1 prevents transcription-associated genomic instability.

R-loops are non-canonical DNA structures that form during transcription and play diverse roles in various physiological processes. Disruption of R-loop homeostasis can lead to genomic instability and replication impairment, contributing to several human diseases, including cancer. Although the molecular mechanisms that protect cells against such events are not fully understood, recent research has identified fork protection factors and DNA damage response proteins as regulators of R-loop dynamics. In this study, we identify the Werner helicase-interacting protein 1 (WRNIP1) as a novel factor that counteracts transcription-associated DNA damage upon replication perturbation. Loss of WRNIP1 leads to R-loop accumulation, resulting in collisions between the replisome and transcription machinery. We observe co-localization of WRNIP1 with transcription/replication complexes and R-loops after replication perturbation, suggesting its involvement in resolving transcription-replication conflicts. Moreover, WRNIP1-deficient cells show impaired replication restart from transcription-induced fork stalling. Notably, transcription inhibition and RNase H1 overexpression rescue all the defects caused by loss of WRNIP1. Importantly, our findings highlight the critical role of WRNIP1 ubiquitin-binding zinc finger (UBZ) domain in preventing pathological persistence of R-loops and limiting DNA damage, thereby safeguarding genome integrity.

RNA polymerase II promotes the organization of chromatin following DNA replication.

Understanding how chromatin organisation is duplicated on the two daughter strands is a central question in epigenetics. In mammals, following the passage of the replisome, nucleosomes lose their defined positioning and transcription contributes to their re-organisation. However, whether transcription plays a greater role in the organization of chromatin following DNA replication remains unclear. Here we analysed protein re-association with newly replicated DNA upon inhibition of transcription using iPOND coupled to quantitative mass spectrometry. We show that nucleosome assembly and the re-establishment of most histone modifications are uncoupled from transcription. However, RNAPII acts to promote the re-association of hundreds of proteins with newly replicated chromatin via pathways that are not observed in steady-state chromatin. These include ATP-dependent remodellers, transcription factors and histone methyltransferases. We also identify a set of DNA repair factors that may handle transcription-replication conflicts during normal transcription in human non-transformed cells. Our study reveals that transcription plays a greater role in the organization of chromatin post-replication than previously anticipated.

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

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.

Rat1 promotes premature transcription termination at R-loops.

Certain DNA sequences can adopt a non-B form in the genome that interfere with DNA-templated processes, including transcription. Among the sequences that are intrinsically difficult to transcribe are those that tend to form R-loops, three-stranded nucleic acid structures formed by a DNA-RNA hybrid and the displaced ssDNA. Here we compared the transcription of an endogenous gene with and without an R-loop-forming sequence inserted. We show that, in agreement with previous in vivo and in vitro analyses, transcription elongation is delayed by R-loops in yeast. Importantly, we demonstrate that the Rat1 transcription terminator factor facilitates transcription throughout such structures by inducing premature termination of arrested RNAPIIs. We propose that RNase H degrades the RNA moiety of the hybrid, providing an entry site for Rat1. Thus, we have uncovered an unanticipated function of Rat1 as a transcription restoring factor opening up the possibility that it may also promote transcription through other genomic DNA structures intrinsically difficult to transcribe. If R-loop-mediated transcriptional stress is not relieved by Rat1, it will cause genomic instability, probably through the increase of transcription-replication conflicts, a deleterious situation that could lead to cancer.