Replication forks stalled at co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage-religation cycles mediated by MUS81 endonuclease and DNA ligase IV (LIG4), which presumably relieve the topological barrier generated by the transcription-replication conflict (TRC) and facilitate ELL-dependent reactivation of transcription. Here, we report that the restart of R-loop-stalled replication forks via the MUS81-LIG4-ELL pathway requires senataxin (SETX), a helicase that can unwind RNA:DNA hybrids. We found that SETX promotes replication fork progression by preventing R-loop accumulation during S-phase. Interestingly, loss of SETX helicase activity leads to nascent DNA degradation upon induction of R-loop-mediated fork stalling by hydroxyurea. This fork degradation phenotype is independent of replication fork reversal and results from DNA2-mediated resection of MUS81-cleaved replication forks that accumulate due to defective replication restart. Finally, we demonstrate that SETX acts in a common pathway with the DEAD-box helicase DDX17 to suppress R-loop-mediated replication stress in human cells. A possible cooperation between these RNA/DNA helicases in R-loop unwinding at TRC sites is discussed.

• MeSH
• Flap Endonucleases metabolism   genetics   MeSH
• DEAD-box RNA Helicases * metabolism   genetics   MeSH
• DNA-Binding Proteins * metabolism   genetics   MeSH
• DNA Helicases * metabolism   genetics   MeSH
• DNA Ligase ATP metabolism   genetics   MeSH
• DNA metabolism   genetics   MeSH
• Endonucleases * metabolism   genetics   MeSH
• Transcription, Genetic MeSH
• Humans MeSH
• Multifunctional Enzymes * metabolism   genetics   MeSH
• R-Loop Structures * MeSH
• DNA Replication * MeSH
• RNA Helicases * metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Flap Endonucleases MeSH
• DEAD-box RNA Helicases * MeSH
• DNA-Binding Proteins * MeSH
• DNA Helicases * MeSH
• DNA Ligase ATP MeSH
• DNA MeSH
• Endonucleases * MeSH
• Multifunctional Enzymes * MeSH
• MUS81 protein, human MeSH Browser
• RNA Helicases * MeSH
• SETX protein, human MeSH Browser

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.

• MeSH
• DNA Helicases genetics   metabolism   MeSH
• DNA genetics   MeSH
• Humans MeSH
• Fanconi Anemia Complementation Group Proteins * genetics   metabolism   MeSH
• R-Loop Structures * MeSH
• DNA Replication MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Helicases MeSH
• DNA MeSH
• Fanconi Anemia Complementation Group Proteins * MeSH

Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.

• MeSH
• DNA-Binding Proteins * metabolism   MeSH
• DNA MeSH
• Hydroxyurea pharmacology   MeSH
• Humans MeSH
• Reactive Oxygen Species MeSH
• DNA Replication * MeSH
• S Phase genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Binding Proteins * MeSH
• DNA MeSH
• Hydroxyurea MeSH
• Reactive Oxygen Species MeSH

R-loops are three-stranded nucleic acid structures composed of an RNA:DNA hybrid and displaced DNA strand. These structures can halt DNA replication when formed co-transcriptionally in the opposite orientation to replication fork progression. A recent study has shown that replication forks stalled by co-transcriptional R-loops can be restarted by a mechanism involving fork cleavage by MUS81 endonuclease, followed by ELL-dependent reactivation of transcription, and fork religation by the DNA ligase IV (LIG4)/XRCC4 complex. However, how R-loops are eliminated to allow the sequential restart of transcription and replication in this pathway remains elusive. Here, we identified the human DDX17 helicase as a factor that associates with R-loops and counteracts R-loop-mediated replication stress to preserve genome stability. We show that DDX17 unwinds R-loops in vitro and promotes MUS81-dependent restart of R-loop-stalled forks in human cells in a manner dependent on its helicase activity. Loss of DDX17 helicase induces accumulation of R-loops and the formation of R-loop-dependent anaphase bridges and micronuclei. These findings establish DDX17 as a component of the MUS81-LIG4-ELL pathway for resolution of R-loop-mediated transcription-replication conflicts, which may be involved in R-loop unwinding.

• MeSH
• DEAD-box RNA Helicases genetics   metabolism   MeSH
• DNA Helicases metabolism   MeSH
• DNA metabolism   MeSH
• Endonucleases metabolism   MeSH
• Humans MeSH
• R-Loop Structures * MeSH
• DNA Replication * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DDX17 protein, human MeSH Browser
• DEAD-box RNA Helicases MeSH
• DNA Helicases MeSH
• DNA MeSH
• Endonucleases MeSH

Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.

• MeSH
• Acetyltransferases genetics   metabolism   MeSH
• HeLa Cells MeSH
• Intracellular Signaling Peptides and Proteins genetics   metabolism   MeSH
• Nuclear Pore Complex Proteins genetics   metabolism   MeSH
• Humans MeSH
• Protein Interaction Maps MeSH
• Neoplasms genetics   MeSH
• Genomic Instability MeSH
• DNA Damage MeSH
• Proto-Oncogene Proteins genetics   metabolism   MeSH
• DNA Replication * MeSH
• RNA Transport MeSH
• Cell Survival MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acetyltransferases MeSH
• Intracellular Signaling Peptides and Proteins MeSH
• Nuclear Pore Complex Proteins MeSH
• MCM3AP protein, human MeSH Browser
• Proto-Oncogene Proteins MeSH
• TPR protein, human MeSH Browser

R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.

• Keywords
• B02 inhibitor, G1 phase of the cell cycle, R-loop, RAD51, origin of replication, pre-replication complex,
• MeSH
• Chromosomal Instability drug effects   MeSH
• DNA biosynthesis   MeSH
• G1 Phase drug effects   MeSH
• Enzyme Inhibitors pharmacology   MeSH
• Origin Recognition Complex metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• R-Loop Structures * MeSH
• Rad51 Recombinase * antagonists & inhibitors   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH
• Enzyme Inhibitors MeSH
• Origin Recognition Complex MeSH
• ORC2 protein, human MeSH Browser
• RAD51 protein, human MeSH Browser
• Rad51 Recombinase * MeSH

Exposure of gastric epithelial cells to the bacterial carcinogen Helicobacter pylori causes DNA double strand breaks. Here, we show that H. pylori-induced DNA damage occurs co-transcriptionally in S-phase cells that activate NF-κB signaling upon innate immune recognition of the lipopolysaccharide biosynthetic intermediate β-ADP-heptose by the ALPK1/TIFA signaling pathway. DNA damage depends on the bi-functional RfaE enzyme and the Cag pathogenicity island of H. pylori, is accompanied by replication fork stalling and can be observed also in primary cells derived from gastric organoids. Importantly, H. pylori-induced replication stress and DNA damage depend on the presence of co-transcriptional RNA/DNA hybrids (R-loops) that form in infected cells during S-phase as a consequence of β-ADP-heptose/ ALPK1/TIFA/NF-κB signaling. H. pylori resides in close proximity to S-phase cells in the gastric mucosa of gastritis patients. Taken together, our results link bacterial infection and NF-κB-driven innate immune responses to R-loop-dependent replication stress and DNA damage.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   metabolism   MeSH
• Bacterial Proteins metabolism   MeSH
• DNA chemistry   genetics   MeSH
• Floxuridine MeSH
• Glycosyltransferases metabolism   MeSH
• Helicobacter pylori metabolism   pathogenicity   MeSH
• Helicobacter Infections metabolism   microbiology   MeSH
• Host-Pathogen Interactions physiology   MeSH
• Humans MeSH
• Lipopolysaccharides metabolism   MeSH
• Mutation MeSH
• Cell Line, Tumor MeSH
• Stomach Neoplasms genetics   microbiology   pathology   MeSH
• NF-kappa B genetics   metabolism   MeSH
• DNA Damage MeSH
• Protein Kinases genetics   metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• DNA Replication drug effects   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• ALPK1 protein, human MeSH Browser
• Bacterial Proteins MeSH
• DNA MeSH
• doxifluridine MeSH Browser
• Floxuridine MeSH
• Glycosyltransferases MeSH
• Lipopolysaccharides MeSH
• NF-kappa B MeSH
• Protein Kinases MeSH
• Reactive Oxygen Species MeSH
• RfaE protein, Bacteria MeSH Browser
• TIFA protein, human MeSH Browser