DNA double-strand breaks (DSBs) represent a lethal form of DNA damage that can trigger cell death or initiate oncogenesis. The activity of RNA polymerase II (RNAPII) at the break site is required for efficient DSB repair. However, the regulatory mechanisms governing the transcription cycle at DSBs are not well understood. Here, we show that Integrator complex subunit 6 (INTS6) associates with the heterotrimeric sensor of ssDNA (SOSS1) complex (comprising INTS3, INIP and hSSB1) to form the tetrameric SOSS1 complex. INTS6 binds to DNA:RNA hybrids and promotes Protein Phosphatase 2A (PP2A) recruitment to DSBs, facilitating the dephosphorylation of RNAPII. Furthermore, INTS6 prevents the accumulation of damage-associated RNA transcripts (DARTs) and the stabilization of DNA:RNA hybrids at DSB sites. INTS6 interacts with and promotes the recruitment of senataxin (SETX) to DSBs, facilitating the resolution of DNA:RNA hybrids/R-loops. Our results underscore the significance of the tetrameric SOSS1 complex in the autoregulation of DNA:RNA hybrids and efficient DNA repair.
- MeSH
- DNA-Binding Proteins metabolism MeSH
- DNA Helicases metabolism genetics MeSH
- DNA * metabolism chemistry MeSH
- DNA Breaks, Double-Stranded * MeSH
- Phosphorylation MeSH
- Homeostasis genetics MeSH
- Humans MeSH
- DNA Repair * MeSH
- Protein Phosphatase 2 metabolism genetics MeSH
- R-Loop Structures MeSH
- RNA Helicases metabolism genetics MeSH
- RNA Polymerase II * metabolism MeSH
- RNA * metabolism genetics chemistry MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article 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
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
BACKGROUND AND PURPOSE: In our previous study, repeated sessions of repetitive transcranial magnetic stimulation (rTMS) over the auditory feedback area were shown to improve hypokinetic dysarthria (HD) in Parkinson's disease (PD) and led to changes in functional connectivity within the left-sided articulatory networks. We analyzed data from this previous study and assessed the effects of rTMS for HD in PD on the diffusion parameters of the left anterior arcuate fasciculus (AAF), which connects the auditory feedback area with motor regions involved in articulation. METHODS: Patients were assigned to 10 sessions of real or sham 1-Hz stimulation over the right posterior superior temporal gyrus. Stimulation effects were evaluated using magnetic resonance diffusion tensor imaging and by a speech therapist using a validated tool (Phonetics score of the Dysarthric Profile) at baseline, immediately after 2 weeks of stimulation, and at follow-up visits at Weeks 6 and 10 after the baseline. RESULTS: Altogether, data from 33 patients were analyzed. A linear mixed model revealed significant time-by-group interaction (p = 0.006) for the relative changes of fractional anisotropy of the AAF; the value increases were associated with the temporal evolution of the Phonetics score (R = 0.367, p = 0.028) in the real stimulation group. CONCLUSIONS: Real rTMS treatment for HD in PD as compared to sham stimulation led to increases of white matter integrity of the auditory-motor loop during the 2-month follow-up period. The changes were related to motor speech improvements.
Double-strand breaks (DSBs) are the most severe type of DNA damage. Previously, we demonstrated that RNA polymerase II (RNAPII) phosphorylated at the tyrosine 1 (Y1P) residue of its C-terminal domain (CTD) generates RNAs at DSBs. However, the regulation of transcription at DSBs remains enigmatic. Here, we show that the damage-activated tyrosine kinase c-Abl phosphorylates hSSB1, enabling its interaction with Y1P RNAPII at DSBs. Furthermore, the trimeric SOSS1 complex, consisting of hSSB1, INTS3, and c9orf80, binds to Y1P RNAPII in response to DNA damage in an R-loop-dependent manner. Specifically, hSSB1, as a part of the trimeric SOSS1 complex, exhibits a strong affinity for R-loops, even in the presence of replication protein A (RPA). Our in vitro and in vivo data reveal that the SOSS1 complex and RNAPII form dynamic liquid-like repair compartments at DSBs. Depletion of the SOSS1 complex impairs DNA repair, underscoring its biological role in the R-loop-dependent DNA damage response.
Non-canonical structures (NCS) refer to the various forms of DNA that differ from the B-conformation described by Watson and Crick. It has been found that these structures are usual components of the genome, actively participating in its essential functions. The present review is focused on the nine kinds of NCS appearing or likely to appear in human ribosomal DNA (rDNA): supercoiling structures, R-loops, G-quadruplexes, i-motifs, DNA triplexes, cruciform structures, DNA bubbles, and A and Z DNA conformations. We discuss the conditions of their generation, including their sequence specificity, distribution within the locus, dynamics, and beneficial and detrimental role in the cell.
- MeSH
- G-Quadruplexes * MeSH
- Nucleic Acid Conformation MeSH
- Humans MeSH
- DNA, Ribosomal genetics MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
INTRODUCTION: T-cell receptor (TCR) recognition of foreign peptides presented by the major histocompatibility complex (MHC) initiates the adaptive immune response against pathogens. While a large number of TCR sequences specific to different antigenic peptides are known to date, the structural data describing the conformation and contacting residues for TCR-peptide-MHC complexes is relatively limited. In the present study we aim to extend and analyze the set of available structures by performing highly accurate template-based modeling of these complexes using TCR sequences with known specificity. METHODS: Identification of CDR3 sequences and their further clustering, based on available spatial structures, V- and J-genes of corresponding T-cell receptors, and epitopes, was performed using the VDJdb database. Modeling of the selected CDR3 loops was conducted using a stepwise introduction of single amino acid substitutions to the template PDB structures, followed by optimization of the TCR-peptide-MHC contacting interface using the Rosetta package applications. Statistical analysis and recursive feature elimination procedures were carried out on computed energy values and properties of contacting amino acid residues between CDR3 loops and peptides, using R. RESULTS: Using the set of 29 complex templates (including a template with SARS-CoV-2 antigen) and 732 specificity records, we built a database of 1585 model structures carrying substitutions in either TCRα or TCRβ chains with some models representing the result of different mutation pathways for the same final structure. This database allowed us to analyze features of amino acid contacts in TCR - peptide interfaces that govern antigen recognition preferences and interpret these interactions in terms of physicochemical properties of interacting residues. CONCLUSION: Our results provide a methodology for creating high-quality TCR-peptide-MHC models for antigens of interest that can be utilized to predict TCR specificity.
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
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
Úvod: Měření práce myokardu (myocardial work, MW) představuje novou echokardiografickou metodu založenou na sledování smyčky tlak-deformace srdeční komory, což umožňuje kvantifikovat výkonnost srdce. Na druhé straně rychlost pulsní vlny (pulse wave velocity, PWV) určuje tuhost tepen na základě poznatku, že se zvyšující se tuhostí tepny se zvyšuje i rychlost anterográdního a retrográdního přenosu sfygmické vlny. Cíl: Cílem této studie bylo stanovit korelaci mezi parametry MW a PWV. Metody: Do studie jsme zařazovali všechny po sobě následující pacienty bez kardiovaskulárního onemocnění, kteří v období mezi červnem 2021 a červencem 2022 absolvovali transtorakální dopplerovské echokardio- grafické vyšetření. Hodnoty MW byly vypočítány ze smyčky tlak-deformace srdeční komory, do níž byly začleněny hodnoty neinvazivního vyšetření tepenného tlaku podle doporučení pro standardní echokardiogracfické vyšetření metodou "speckle tracking". Hodnota PWV se měřila tonometrem na úrovni společné karotidy a společné femorální tepny. Výsledky: Celkem bylo do studie zařazeno 66 pacientů průměrného věku 30,7 ± 8,6 roku. Byla nalezena statisticky významná negativní korelace mezi PWV a celkovou zbytečně vynaloženou energií (global wasted energy, GWE) (r = -0,317; p < 0,01) při korelaci s celkovou zbytečně vynaloženou prací (global wasted work, GWW) (r = 0,324; p < 0,01). Statisticky významná korelace přetrvávala v podskupinách žen i mužů u GWE (ženy: r = -0,280; p < 0,05; muži: r = -0,362; p < 0,05) i u GWW (ženy: r = 0,359; p < 0,05; muži: r = 0,359; p < 0,05). Závěr: Vztah mezi MW a PWV jako projev součinnosti mezi levou komorou a velkými tepnami může potenciálně představovat užitečný nástroj pro časné odhalení subklinické dysfunkce kardiovaskulárního systému.
Introduction: The myocardial work (MW) is a new echocardiographic method, based on the pressure-strain loop, which allows quantifying the cardiac performance. On the other hand, the pulse wave velocity (PWV) evaluates arterial stiffness, knowing that as the stiffness of an artery increases, the transmission velocity of the anterograde and the retrograde sphygmic wave increases. Purpose: The aim of the study is to evaluate the correlation between MW and PWV parameters. Methods: We enrolled consecutively all patients without cardiovascular disease who underwent transthora- cic Doppler echocardiography between June 2021 and July 2022. The MW parameters were derived from the strain-pressure loop, including in its calculation the measurement non-invasive arterial pressure, according to standard speckle tracking echocardiography recommendations. The PWV measurement was obtained by tonometry at the level of the common carotid artery and the common femoral artery. Results: We enrolled 66 patients (mean age: 30.7±8.6 years). There was a significant inversely proportio- nal correlation between PWV and GWE (r = –0.317; p <0.01) meanwhile there was a directly proportional correlation with GWW (r = 0.324; p <0.01). The statistically significant correlation remained in the female and male subgroups for GWE (female: r = –0.280; p <0.05; male: r = –0.362; p <0.05) and GWW (female: r = 0.359; p <0.05; male: r = 0.359; p <0.05). Conclusion: The relationship between MW and PWV as a demonstration of ventricular arterial coupling may potentially be a useful tool in the early recognition of subclinical cardiovascular dysfunction.
- MeSH
- Pulse Wave Analysis * methods instrumentation MeSH
- Echocardiography, Doppler methods instrumentation MeSH
- Adult MeSH
- Correlation of Data MeSH
- Humans MeSH
- Young Adult MeSH
- Heart * physiology MeSH
- Statistics as Topic MeSH
- Vascular Stiffness physiology MeSH
- Check Tag
- Adult MeSH
- Humans MeSH
- Young Adult MeSH
- Male MeSH
- Female MeSH
- Publication type
- Clinical Study MeSH
- Geographicals
- Italy MeSH
