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" endonukleasy metabolismus   genetika   MeSH
• DEAD-box RNA-helikasy * metabolismus   genetika   MeSH
• DNA vazebné proteiny * metabolismus   genetika   MeSH
• DNA-helikasy * metabolismus   genetika   MeSH
• DNA-ligasa ATP metabolismus   genetika   MeSH
• DNA metabolismus   genetika   MeSH
• endonukleasy * metabolismus   genetika   MeSH
• genetická transkripce MeSH
• lidé MeSH
• multifunkční enzymy * metabolismus   genetika   MeSH
• R-smyčka * MeSH
• replikace DNA * MeSH
• RNA-helikasy * metabolismus   genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• "flap" endonukleasy MeSH
• DEAD-box RNA-helikasy * MeSH
• DNA vazebné proteiny * MeSH
• DNA-helikasy * MeSH
• DNA-ligasa ATP MeSH
• DNA MeSH
• endonukleasy * MeSH
• multifunkční enzymy * MeSH
• MUS81 protein, human MeSH Prohlížeč
• RNA-helikasy * MeSH
• SETX protein, human MeSH Prohlížeč

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-helikasy genetika   metabolismus   MeSH
• DNA genetika   MeSH
• lidé MeSH
• proteiny FANC * genetika   metabolismus   MeSH
• R-smyčka * MeSH
• replikace DNA MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA-helikasy MeSH
• DNA MeSH
• proteiny FANC * MeSH

Helicobacter pylori infection is a major risk factor for the development of gastric cancer. The bacteria reside in close proximity to gastric surface mucous as well as stem and progenitor cells. Here, we take advantage of wild-type and genetically engineered murine gastric organoids and organoid-derived monolayers to study the cellular targets of H. pylori-induced DNA damage and replication stress and to explore possible interactions with preexisting gastric cancer driver mutations. We find using alkaline comet assay, single-molecule DNA fiber assays, and immunofluorescence microscopy of DNA repair foci that H. pylori induces transcription-dependent DNA damage in actively replicating, Leucine-rich-repeat containing G-Protein-Coupled Receptor 5 (Lgr5)-positive antral stem and progenitor cells and their Troy-positive corpus counterparts, but not in other gastric epithelial lineages. Infection-dependent DNA damage is aggravated by Apc inactivation, but not by Trp53 or Smad4 loss, or Erbb2 overexpression. Our data suggest that H. pylori induces DNA damage in stem and progenitor cells, especially in settings of hyperproliferation due to constitutively active Wnt signaling.

• MeSH
• Helicobacter pylori * genetika   MeSH
• infekce vyvolané Helicobacter pylori * genetika   mikrobiologie   MeSH
• kmenové buňky MeSH
• lidé MeSH
• myši MeSH
• nádory žaludku * patologie   MeSH
• poškození DNA MeSH
• receptory spřažené s G-proteiny genetika   MeSH
• tumor supresorové geny MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adenomatous polyposis coli protein, mouse MeSH Prohlížeč
• receptory spřažené s G-proteiny MeSH

Guanine-quadruplex structures (G4) are unusual nucleic acid conformations formed by guanine-rich DNA and RNA sequences and known to control gene expression mechanisms, from transcription to protein synthesis. So far, a number of molecules that recognize G4 have been developed for potential therapeutic applications in human pathologies, including cancer and infectious diseases. These molecules are called G4 ligands. When the biological effects of G4 ligands are studied, the analysis is often limited to nucleic acid targets. However, recent evidence indicates that G4 ligands may target other cellular components and compartments such as lysosomes and mitochondria. Here, we summarize our current knowledge of the regulation of lysosome by G4 ligands, underlying their potential functional impact on lysosome biology and autophagic flux, as well as on the transcriptional regulation of lysosomal genes. We outline the consequences of these effects on cell fate decisions and we systematically analyzed G4-prone sequences within the promoter of 435 lysosome-related genes. Finally, we propose some hypotheses about the mechanisms involved in the regulation of lysosomes by G4 ligands.

• Klíčová slova
• Autophagy, TFEB, guanine-quadruplex, lysosome membrane permeabilization, transcriptional regulation,
• MeSH
• autofagie * MeSH
• DNA metabolismus   MeSH
• G-kvadruplexy * MeSH
• guanin MeSH
• lidé MeSH
• ligandy MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH
• guanin MeSH
• ligandy 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 vazebné proteiny * metabolismus   MeSH
• DNA MeSH
• hydroxymočovina farmakologie   MeSH
• lidé MeSH
• reaktivní formy kyslíku MeSH
• replikace DNA * MeSH
• S fáze genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA vazebné proteiny * MeSH
• DNA MeSH
• hydroxymočovina MeSH
• reaktivní formy kyslíku MeSH

The current monkeypox virus (MPXV) strain differs from the strain arising in 2018 by 50+ single nucleotide polymorphisms (SNPs) and is mutating much faster than expected. The cytidine deaminase apolipoprotein B messenger RNA editing enzyme, catalytic subunit B (APOBEC3) was hypothesized to be driving this increased mutation. APOBEC has recently been identified to preferentially mutate cruciform DNA secondary structures formed by inverted repeats (IRs). IRs were recently identified as hot spots for mutation in severe acute respiratory syndrome coronavirus 2, and we aimed to identify whether IRs were also hot spots for mutation within MPXV genomes. We found that MPXV genomes were replete with IR sequences. Of the 50+ SNPs identified in the 2022 outbreak strain, 63.9% of these were found to have arisen within IR regions in the 2018 reference strain (MT903344.1). Notably, IR sequences found in the 2018 reference strain were significantly lost over time, with an average of 32.5% of these sequences being conserved in the 2022 MPXV genomes. This evidence was highly indicative that mutations were arising within IRs. This data provides further support to the hypothesis that APOBEC may be driving MPXV mutation and highlights the necessity for greater surveillance of IRs of MPXV genomes to detect new mutations.

• Klíčová slova
• APOBEC, evolution, inverted repeats, monkeypox, mutation,
• MeSH
• COVID-19 * MeSH
• lidé MeSH
• mutace MeSH
• SARS-CoV-2 MeSH
• virus opičích neštovic * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

Human ribosomal DNA is represented by hundreds of repeats in each cell. Every repeat consists of two parts: a 13 kb long 47S DNA with genes encoding 18S, 5.8S, and 28S RNAs of ribosomal particles, and a 30 kb long intergenic spacer (IGS). Remarkably, transcription does not take place in all the repeats. The transcriptionally silent genes are characterized by the epigenetic marks of the inactive chromatin, including DNA hypermethylation of the promoter and adjacent areas. However, it is still unknown what causes the differentiation of the genes into active and silent. In this study, we examine whether this differentiation is related to the nucleotide sequence of IGS. We isolated ribosomal DNA from the nucleoli of human-derived HT1080 cells, and separated methylated and non-methylated DNA by chromatin immunoprecipitation. Then, we used PCR to amplify a 2 kb long region upstream of the transcription start and sequenced the product. We found that six SNVs and a series of short deletions in a region of simple repeats correlated with the DNA methylation status. These data indicate that variability of IGS sequence may initiate silencing of the ribosomal genes. Our study also suggests a number of pathways to this silencing that involve micro-RNAs and/or non-canonical DNA structures.

• Klíčová slova
• micro-RNAs, non-canonical DNA structures, rDNA, sequence variability, transcription,
• MeSH
• intergenová DNA MeSH
• lidé MeSH
• mezerníky ribozomální DNA genetika   MeSH
• ribozomální DNA genetika   MeSH
• ribozomy * MeSH
• RNA ribozomální 28S genetika   MeSH
• sekvence nukleotidů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• intergenová DNA MeSH
• mezerníky ribozomální DNA MeSH
• ribozomální DNA MeSH
• RNA ribozomální 28S 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-helikasy genetika   metabolismus   MeSH
• DNA-helikasy metabolismus   MeSH
• DNA metabolismus   MeSH
• endonukleasy metabolismus   MeSH
• lidé MeSH
• R-smyčka * MeSH
• replikace DNA * genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DDX17 protein, human MeSH Prohlížeč
• DEAD-box RNA-helikasy MeSH
• DNA-helikasy MeSH
• DNA MeSH
• endonukleasy 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.

• Klíčová slova
• B02 inhibitor, G1 phase of the cell cycle, R-loop, RAD51, origin of replication, pre-replication complex,
• MeSH
• chromozomální nestabilita účinky léků   MeSH
• DNA biosyntéza   MeSH
• G1 fáze účinky léků   MeSH
• inhibitory enzymů farmakologie   MeSH
• komplex rozpoznávající replikační počátek metabolismus   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• R-smyčka * MeSH
• rekombinasa Rad51 * antagonisté a inhibitory   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• DNA MeSH
• inhibitory enzymů MeSH
• komplex rozpoznávající replikační počátek MeSH
• ORC2 protein, human MeSH Prohlížeč
• RAD51 protein, human MeSH Prohlížeč
• rekombinasa Rad51 * MeSH

The ends of eukaryotic chromosomes typically contain a 3' ssDNA G-rich protrusion (G-overhang). This overhang must be protected against detrimental activities of nucleases and of the DNA damage response machinery and participates in the regulation of telomerase, a ribonucleoprotein complex that maintains telomere integrity. These functions are mediated by DNA-binding proteins, such as Cdc13 in Saccharomyces cerevisiae, and the propensity of G-rich sequences to form various non-B DNA structures. Using CD and NMR spectroscopies, we show here that G-overhangs of S. cerevisiae form distinct Hoogsteen pairing-based secondary structures, depending on their length. Whereas short telomeric oligonucleotides form a G-hairpin, their longer counterparts form parallel and/or antiparallel G-quadruplexes (G4s). Regardless of their topologies, non-B DNA structures exhibited impaired binding to Cdc13 in vitro as demonstrated by electrophoretic mobility shift assays. Importantly, whereas G4 structures formed relatively quickly, G-hairpins folded extremely slowly, indicating that short G-overhangs, which are typical for most of the cell cycle, are present predominantly as single-stranded oligonucleotides and are suitable substrates for Cdc13. Using ChIP, we show that the occurrence of G4 structures peaks at the late S phase, thus correlating with the accumulation of long G-overhangs. We present a model of how time- and length-dependent formation of non-B DNA structures at chromosomal termini participates in telomere maintenance.

• Klíčová slova
• Cdc13, G-hairpin, G-quadruplex, Saccharomyces cerevisiae, cell cycle, folding kinetics, telomerase, telomere,
• MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA metabolismus   MeSH
• G-kvadruplexy MeSH
• homeostáza telomer fyziologie   MeSH
• jednovláknová DNA metabolismus   MeSH
• kinetika MeSH
• konformace nukleové kyseliny MeSH
• oligonukleotidy genetika   MeSH
• proteiny vázající telomery metabolismus   MeSH
• retardační test MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• telomerasa genetika   MeSH
• telomery metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• Cdc13 protein, S cerevisiae MeSH Prohlížeč
• DNA vazebné proteiny MeSH
• DNA MeSH
• jednovláknová DNA MeSH
• oligonukleotidy MeSH
• proteiny vázající telomery MeSH
• Saccharomyces cerevisiae - proteiny MeSH
• telomerasa MeSH