R-loops are common non-B nucleic acid structures formed by a three-stranded nucleic acid composed of an RNA-DNA hybrid and a displaced single-stranded DNA (ssDNA) loop. Because the aberrant R-loop formation leads to increased mutagenesis, hyper-recombination, rearrangements, and transcription-replication collisions, it is regarded as important in human diseases. Therefore, its prevalence and distribution in genomes are studied intensively. However, in silico tools for R-loop prediction are limited, and therefore, we have developed the R-loop tracker tool, which was implemented as a part of the DNA Analyser web server. This new tool is focused upon (1) prediction of R-loops in genomic DNA without length and sequence limitations; (2) integration of R-loop tracker results with other tools for nucleic acids analyses, including Genome Browser; (3) internal cross-evaluation of in silico results with experimental data, where available; (4) easy export and correlation analyses with other genome features and markers; and (5) enhanced visualization outputs. Our new R-loop tracker tool is freely accessible on the web pages of DNA Analyser tools, and its implementation on the web-based server allows effective analyses not only for DNA segments but also for full chromosomes and genomes.

• Keywords
• RNA–DNA hybrid, non-B structure, sequence analysis,
• MeSH
• Algorithms * MeSH
• DNA chemistry   genetics   MeSH
• Genomics methods   MeSH
• Internet statistics & numerical data   MeSH
• Humans MeSH
• Genomic Instability * MeSH
• R-Loop Structures * MeSH
• Software MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA MeSH

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

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

Prolonged pausing of the transcription machinery may lead to the formation of three-stranded nucleic acid structures, called R-loops, typically resulting from the annealing of the nascent RNA with the template DNA. Unscheduled persistence of R-loops and RNA polymerases may interfere with transcription itself and other essential processes such as DNA replication and repair. Senataxin (SETX) is a putative helicase, mutated in two neurodegenerative disorders, which has been implicated in the control of R-loop accumulation and in transcription termination. However, understanding the precise role of SETX in these processes has been precluded by the absence of a direct characterisation of SETX biochemical activities. Here, we purify and characterise the helicase domain of SETX in parallel with its yeast orthologue, Sen1. Importantly, we show that SETX is a bona fide helicase with the ability to resolve R-loops. Furthermore, SETX has retained the transcription termination activity of Sen1 but functions in a species-specific manner. Finally, subsequent characterisation of two SETX variants harbouring disease-associated mutations shed light into the effect of such mutations on SETX folding and biochemical properties. Altogether, these results broaden our understanding of SETX function in gene expression and the maintenance of genome integrity and provide clues to elucidate the molecular basis of SETX-associated neurodegenerative diseases.

• MeSH
• DNA Helicases * genetics   metabolism   MeSH
• Transcription, Genetic MeSH
• Humans MeSH
• Multifunctional Enzymes genetics   metabolism   MeSH
• Neurodegenerative Diseases MeSH
• R-Loop Structures MeSH
• Gene Expression Regulation MeSH
• RNA Helicases * metabolism   MeSH
• Saccharomyces cerevisiae Proteins metabolism   MeSH
• Saccharomyces cerevisiae metabolism   MeSH
• Transcription Termination, Genetic * MeSH
• Transcription Factors genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA Helicases * MeSH
• Multifunctional Enzymes MeSH
• RNA Helicases * MeSH
• Saccharomyces cerevisiae Proteins MeSH
• SEN1 protein, S cerevisiae MeSH Browser
• SETX protein, human MeSH Browser
• Transcription Factors MeSH

Up to 15% of human cancers maintain their telomeres through a telomerase-independent mechanism, termed "alternative lengthening of telomeres" (ALT) that relies on homologous recombination between telomeric sequences. Emerging evidence suggests that the recombinogenic nature of ALT telomeres results from the formation of RNA:DNA hybrids (R-loops) between telomeric DNA and the long-noncoding telomeric repeat-containing RNA (TERRA). Here, we show that the mismatch repair protein MutSβ, a heterodimer of MSH2 and MSH3 subunits, is enriched at telomeres in ALT cancer cells, where it prevents the accumulation of telomeric G-quadruplex (G4) structures and R-loops. Cells depleted of MSH3 display increased incidence of R-loop-dependent telomere fragility and accumulation of telomeric C-circles. We also demonstrate that purified MutSβ recognizes and destabilizes G4 structures in vitro. These data suggest that MutSβ destabilizes G4 structures in ALT telomeres to regulate TERRA R-loops, which is a prerequisite for maintenance of telomere integrity during ALT.

• Keywords
• ALT cancers, C-circle, CP: Cancer, CP: Molecular biology, G-quadruplex, R-loop, mismatch repair, telomere,
• MeSH
• DNA metabolism   MeSH
• Telomere Homeostasis MeSH
• Humans MeSH
• Neoplasms * genetics   MeSH
• R-Loop Structures MeSH
• RNA, Long Noncoding * metabolism   MeSH
• Telomere metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA MeSH
• RNA, Long Noncoding * MeSH

Telomeres-repeated, noncoding nucleotide motifs and associated proteins that are found at the ends of eukaryotic chromosomes-mediate genome stability and determine cellular lifespan1. Telomeric-repeat-containing RNA (TERRA) is a class of long noncoding RNAs (lncRNAs) that are transcribed from chromosome ends2,3; these RNAs in turn regulate telomeric chromatin structure and telomere maintenance through the telomere-extending enzyme telomerase4-6 and homology-directed DNA repair7,8. The mechanisms by which TERRA is recruited to chromosome ends remain poorly defined. Here we develop a reporter system with which to dissect the underlying mechanisms, and show that the UUAGGG repeats of TERRA are both necessary and sufficient to target TERRA to chromosome ends. TERRA preferentially associates with short telomeres through the formation of telomeric DNA-RNA hybrid (R-loop) structures that can form in trans. Telomere association and R-loop formation trigger telomere fragility and are promoted by the recombinase RAD51 and its interacting partner BRCA2, but counteracted by the RNA-surveillance factors RNaseH1 and TRF1. RAD51 physically interacts with TERRA and catalyses R-loop formation with TERRA in vitro, suggesting a direct involvement of this DNA recombinase in the recruitment of TERRA by strand invasion. Together, our findings reveal a RAD51-dependent pathway that governs TERRA-mediated R-loop formation after transcription, providing a mechanism for the recruitment of lncRNAs to new loci in trans.

• MeSH
• Biocatalysis MeSH
• HeLa Cells MeSH
• Humans MeSH
• Telomeric Repeat Binding Protein 1 metabolism   MeSH
• R-Loop Structures * MeSH
• Rad51 Recombinase metabolism   MeSH
• Genes, Reporter MeSH
• Ribonuclease H metabolism   MeSH
• RNA, Long Noncoding chemistry   genetics   MeSH
• Base Sequence MeSH
• Telomere chemistry   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Telomeric Repeat Binding Protein 1 MeSH
• RAD51 protein, human MeSH Browser
• Rad51 Recombinase MeSH
• ribonuclease HI MeSH Browser
• Ribonuclease H MeSH
• RNA, Long Noncoding MeSH

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.

• Keywords
• DNA ligase IV, MUS81, R-loop, RECQ5, replication fork reversal, replication restart, replication stress, transcription-replication conflict,
• MeSH
• Rad52 DNA Repair and Recombination Protein metabolism   MeSH
• DNA-Binding Proteins metabolism   MeSH
• DNA Ligases metabolism   MeSH
• DNA Polymerase III metabolism   MeSH
• Endodeoxyribonucleases metabolism   MeSH
• Endonucleases genetics   metabolism   MeSH
• Transcription, Genetic genetics   MeSH
• HeLa Cells MeSH
• RecQ Helicases metabolism   physiology   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• R-Loop Structures genetics   physiology   MeSH
• Rad51 Recombinase genetics   metabolism   physiology   MeSH
• DNA Replication genetics   physiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Rad52 DNA Repair and Recombination Protein MeSH
• DNA-Binding Proteins MeSH
• DNA Ligases MeSH
• DNA Polymerase III MeSH
• Endodeoxyribonucleases MeSH
• Endonucleases MeSH
• RecQ Helicases MeSH
• Lig4 protein, Arabidopsis MeSH Browser
• MUS81 protein, human MeSH Browser
• RAD51 protein, human MeSH Browser
• RAD52 protein, human MeSH Browser
• RECQL protein, human MeSH Browser
• RECQL5 protein, human MeSH Browser
• Rad51 Recombinase MeSH
• XRCC4 protein, human MeSH Browser

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

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

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