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
Cíl: Zjistit míru metylací DNA vybraných genových promotorů u jednotlivých typů hyperplazie endometria ve srovnání s normální endometriální tkání. Soubor a metodika: Byla použita MS-MLPA (multiplex ligation-dependent probe amplification). Porovnáno bylo celkem 120 vzorků tkáně endometria; 40 vzorků s atypickou hyperplazií endometria, 40 vzorků s hyperplazií endometria bez atypií a 40 kontrolních vzorků tkáně zdravého endometria. Výsledky a závěry: Rozdíly v metylaci DNA mezi jednotlivými skupinami byly zjištěny v genech TWIST1, GATA4, MUS81 a NTRK1 (TWIST1: atypická hyperplazie 67,5 %, benigní hyperplazie 2,5 %, normální endometrium 22,5 %; p < 0,00001; GATA4: atypická hyperplazie 95,0 %, benigní hyperplazie 65,0 %, normální endometrium 22,5 %; p < 0,00001; MUS81: atypická hyperplazie 57,5 %, benigní hyperplazie 22,5 %, normální endo-metrium 5,0 %; p < 0,00001; NTRK1: atypická hyperplazie 65,0 %, benigní hyperplazie 27,5 %, normální endometrium 10 %; p < 0,00001). U genů TWIST1, GATA4, MUS81 a NTRK1 byla pozorována vyšší míra metylace u vzorků s atypickou hyperplazií endometria v porovnání se vzorky zdravého endometria a dále byla vyšší míra metylace pozorována u vzorků s atypickou hyperplazií endometria v porovnání se vzorky benigní hyperplazie endometria. Metylace DNA u tumor supresorových genů TWIST1, GATA4, MUS81 a NTRK1 se uplatňuje v patogenezi atypické hyperplazie endometria.
Objective: To investigate DNA methylation of specific tumor suppressor genes in endometrial hyperplasia compared to normal endometrial tissue. File and methodology: To search for epigenetic events, methylation-specific multiplex ligation-dependent probe amplification was employed to compare the methylation status of 40 tissue samples with atypical endometrial hyperplasia, 40 tissue samples with endometrial hyperplasia without atypia, and 40 control tissue samples with a normal endometrium. Results and conclusion: Differences in DNA methylation among the groups were found in TWIST1, GATA4, MUS81, and NTRK1 genes (TWIST1: atypical hyperplasia 67.5%, benign hyperplasia 2.5%, normal endometrium 22.5%; P < 0.00001; GATA4: atypical hyperplasia 95%, benign hyperplasia 65%, normal endometrium 22.5%; P < 0.00001; MUS81: atypical hyperplasia 57.5%, benign hyperplasia 22.5%, normal endometrium 5%; P < 0.00001; NTRK1: atypical hyperplasia 65%, benign hyperplasia 27.5%, normal endometrium 10%; P < 0.00001). Higher methylation rates were observed for the tumor suppressor genes of TWIST1, GATA4, MUS81, and NTRK1 in samples with atypical endometrial hyperplasia compared to samples with normal endometrial tissue, and higher methylation rates were found in samples with atypical endometrial hyperplasia compared to samples of benign endometrial hyperplasia. DNA methylation of TWIST1, GATA4, MUS81, and NTRK1 is involved in the pathogenesis of atypical endometrial hyperplasia.
- MeSH
- DNA vazebné proteiny genetika MeSH
- endonukleasy genetika MeSH
- hyperplazie endometria * genetika metabolismus patologie MeSH
- jaderné proteiny genetika MeSH
- klinická studie jako téma MeSH
- lidé MeSH
- metylace DNA MeSH
- receptor trkA genetika MeSH
- transkripční faktor GATA4 genetika metabolismus MeSH
- transkripční faktor Twist genetika MeSH
- tumor supresorové geny MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
In mammals, RNA interference (RNAi) was historically studied as a cytoplasmic event; however, in the last decade, a growing number of reports convincingly show the nuclear localization of the Argonaute (AGO) proteins. Nevertheless, the extent of nuclear RNAi and its implication in biological mechanisms remain to be elucidated. We found that reduced Lamin A levels significantly induce nuclear influx of AGO2 in SHSY5Y neuroblastoma and A375 melanoma cancer cell lines, which normally have no nuclear AGO2. Lamin A KO manifested a more pronounced effect in SHSY5Y cells compared to A375 cells, evident by changes in cell morphology, increased cell proliferation, and oncogenic miRNA expression. Moreover, AGO fPAR-CLIP in Lamin A KO SHSY5Y cells revealed significantly reduced RNAi activity. Further exploration of the nuclear AGO interactome by mass spectrometry identified FAM120A, an RNA-binding protein and known interactor of AGO2. Subsequent FAM120A fPAR-CLIP, revealed that FAM120A co-binds AGO targets and that this competition reduces the RNAi activity. Therefore, loss of Lamin A triggers nuclear AGO2 translocation, FAM120A mediated RNAi impairment, and upregulation of oncogenic miRNAs, facilitating cancer cell proliferation.
- MeSH
- aktivní transport - buněčné jádro MeSH
- Argonaut proteiny * metabolismus genetika MeSH
- buněčné jádro * metabolismus MeSH
- lamin typ A * metabolismus genetika MeSH
- lidé MeSH
- melanom genetika metabolismus patologie MeSH
- mikro RNA * metabolismus genetika MeSH
- nádorové buněčné linie MeSH
- proliferace buněk * genetika MeSH
- proteiny vázající RNA metabolismus genetika MeSH
- RNA interference * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Homologous recombination involves the formation of branched DNA molecules that may interfere with chromosome segregation. To resolve these persistent joint molecules, cells rely on the activation of structure-selective endonucleases (SSEs) during the late stages of the cell cycle. However, the premature activation of SSEs compromises genome integrity, due to untimely processing of replication and/or recombination intermediates. Here, we used a biochemical approach to show that the budding yeast SSEs Mus81 and Yen1 possess the ability to cleave the central recombination intermediate known as the displacement loop or D-loop. Moreover, we demonstrate that, consistently with previous genetic data, the simultaneous action of Mus81 and Yen1, followed by ligation, is sufficient to recreate the formation of a half-crossover precursor in vitro. Our results provide not only mechanistic explanation for the formation of a half-crossover, but also highlight the critical importance for precise regulation of these SSEs to prevent chromosomal rearrangements.
- MeSH
- crossing over (genetika) * MeSH
- DNA vazebné proteiny * metabolismus genetika MeSH
- endonukleasy * metabolismus genetika MeSH
- homologní rekombinace MeSH
- resolvasy Hollidayova spoje metabolismus genetika MeSH
- Saccharomyces cerevisiae - proteiny * metabolismus genetika MeSH
- Saccharomyces cerevisiae genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
The MRE11, RAD50, and NBN genes encode the MRN complex sensing DNA breaks and directing their repair. While carriers of biallelic germline pathogenic variants (gPV) develop rare chromosomal instability syndromes, the cancer risk in heterozygotes remains controversial. We performed a systematic review and meta-analysis of 53 studies in patients with different cancer diagnoses to better understand the cancer risk. We found an increased risk (odds ratio, 95% confidence interval) for gPV carriers in NBN for melanoma (7.14; 3.30-15.43), pancreatic cancer (4.03; 2.14-7.58), hematological tumors (3.42; 1.14-10.22), and prostate cancer (2.44, 1.84-3.24), but a low risk for breast cancer (1.29; 1.00-1.66) and an insignificant risk for ovarian cancer (1.53; 0.76-3.09). We found no increased breast cancer risk in carriers of gPV in RAD50 (0.93; 0.74-1.16; except of c.687del carriers) and MRE11 (0.87; 0.66-1.13). The secondary burden analysis compared the frequencies of gPV in MRN genes in patients from 150 studies with those in the gnomAD database. In NBN gPV carriers, this analysis additionally showed a high risk for brain tumors (5.06; 2.39-9.52), a low risk for colorectal (1.64; 1.26-2.10) and hepatobiliary (2.16; 1.02-4.06) cancers, and no risk for endometrial, and gastric cancer. The secondary burden analysis showed also a moderate risk for ovarian cancer (3.00; 1.27-6.08) in MRE11 gPV carriers, and no risk for ovarian and hepatobiliary cancers in RAD50 gPV carriers. These findings provide a robust clinical evidence of cancer risks to guide personalized clinical management in heterozygous carriers of gPV in the MRE11, RAD50, and NBN genes.
- MeSH
- DNA vazebné proteiny genetika MeSH
- enzymy opravy DNA genetika MeSH
- genetická predispozice k nemoci * MeSH
- homologní protein MRE11 * genetika MeSH
- hydrolasy působící na anhydridy kyselin * genetika MeSH
- jaderné proteiny * genetika MeSH
- lidé MeSH
- nádory * genetika MeSH
- proteiny buněčného cyklu * genetika MeSH
- zárodečné mutace * MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- metaanalýza MeSH
- systematický přehled MeSH
Meiotic recombination is of central importance for the proper segregation of homologous chromosomes, but also for creating genetic diversity. It is initiated by the formation of double-strand breaks (DSBs) in DNA catalysed by evolutionarily conserved Spo11, together with additional protein partners. Difficulties in purifying the Spo11 protein have limited the characterization of its biochemical properties and of its interactions with other DSB proteins. In this study, we have purified fragments of Spo11 and show for the first time that Spo11 can physically interact with Mre11 and modulates its DNA binding, bridging, and nuclease activities. The interaction of Mre11 with Spo11 requires its far C-terminal region, which is in line with the severe meiotic phenotypes of various mre11 mutations located at the C-terminus. Moreover, calibrated ChIP for Mre11 shows that Spo11 promotes Mre11 recruitment to chromatin, independent of DSB formation. A mutant deficient in Spo11 interaction severely reduces the association of Mre11 with meiotic chromatin. Consistent with the reduction of Mre11 foci in this mutant, it strongly impedes DSB formation, leading to spore death. Our data provide evidence that physical interaction between Spo11 and Mre11, together with end-bridging, promote normal recruitment of Mre11 to hotspots and DSB formation.
- MeSH
- chromatin * metabolismus MeSH
- DNA vazebné proteiny metabolismus genetika MeSH
- dvouřetězcové zlomy DNA * MeSH
- endodeoxyribonukleasy * metabolismus genetika MeSH
- exodeoxyribonukleasy metabolismus genetika MeSH
- meióza * genetika MeSH
- mutace MeSH
- Saccharomyces cerevisiae - proteiny * metabolismus genetika MeSH
- Saccharomyces cerevisiae cytologie genetika metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The World Health Organization (WHO) identifies several bunyaviruses as significant threats to global public health security. Developing effective therapies against these viruses is crucial to combat future outbreaks and mitigate their impact on patient outcomes. Here, we report the synthesis of some isoindol-1-one derivatives and explore their inhibitory properties over an indispensable metal-dependent cap-snatching endonuclease (Cap-ENDO) shared among evolutionary divergent bunyaviruses. The compounds suppressed RNA hydrolysis by Cap-ENDOs, with IC50 values predominantly in the lower μM range. Molecular docking studies revealed the interactions with metal ions to be essential for the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold activity. Calorimetric analysis uncovered Mn2+ ions to have the highest affinity for sites within the targets, irrespective of aminoacidic variations influencing metal cofactor preferences. Interestingly, spectrophotometric findings unveiled sole dinuclear species formation between the scaffold and Mn2+. Moreover, the complexation of two Mn2+ ions within the viral enzymes appears to be favourable, as indicated by the binding of compound 11 to TOSV Cap-ENDO (Kd = 28 ± 3 μM). Additionally, the tendency of compound 11 to stabilize His+ more than His- Cap-ENDOs suggests exploitable differences in their catalytic pockets relevant to improving specificity. Collectively, our results underscore the isoindolinone scaffold's potential as a strategic starting point for the design of pan-antibunyavirus drugs.
- MeSH
- antivirové látky farmakologie chemie chemická syntéza MeSH
- Bunyaviridae účinky léků metabolismus MeSH
- endonukleasy * metabolismus antagonisté a inhibitory MeSH
- inhibitory enzymů farmakologie chemie chemická syntéza MeSH
- isoindoly chemická syntéza farmakologie chemie MeSH
- lidé MeSH
- molekulární struktura MeSH
- racionální návrh léčiv * MeSH
- simulace molekulového dockingu * MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Targeting poly(ADP-ribose) glycohydrolase (PARG) is currently explored as a therapeutic approach to treat various cancer types, but we have a poor understanding of the specific genetic vulnerabilities that would make cancer cells susceptible to such a tailored therapy. Moreover, the identification of such vulnerabilities is of interest for targeting BRCA2;p53-deficient tumors that have acquired resistance to poly(ADP-ribose) polymerase inhibitors (PARPi) through loss of PARG expression. Here, by performing whole-genome CRISPR/Cas9 drop-out screens, we identify various genes involved in DNA repair to be essential for the survival of PARG;BRCA2;p53-deficient cells. In particular, our findings reveal EXO1 and FEN1 as major synthetic lethal interactors of PARG loss. We provide evidence for compromised replication fork progression, DNA single-strand break repair, and Okazaki fragment processing in PARG;BRCA2;p53-deficient cells, alterations that exacerbate the effects of EXO1/FEN1 inhibition and become lethal in this context. Since this sensitivity is dependent on BRCA2 defects, we propose to target EXO1/FEN1 in PARPi-resistant tumors that have lost PARG activity. Moreover, EXO1/FEN1 targeting may be a useful strategy for enhancing the effect of PARG inhibitors in homologous recombination-deficient tumors.
- MeSH
- "flap" endonukleasy genetika metabolismus terapeutické užití MeSH
- enzymy opravy DNA genetika MeSH
- exodeoxyribonukleasy genetika MeSH
- glykosidhydrolasy genetika metabolismus MeSH
- lidé MeSH
- nádorový supresorový protein p53 * genetika metabolismus MeSH
- nádory * farmakoterapie genetika MeSH
- oprava DNA MeSH
- PARP inhibitory farmakologie MeSH
- poškození DNA MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts.
ISG20L2, a 3' to 5' exoribonuclease previously associated with ribosome biogenesis, is identified here in activated T cells as an enzyme with a preferential affinity for uridylated miRNA substrates. This enzyme is upregulated in T lymphocytes upon TCR and IFN type I stimulation and appears to be involved in regulating T cell function. ISG20L2 silencing leads to an increased basal expression of CD69 and induces greater IL2 secretion. However, ISG20L2 absence impairs CD25 upregulation, CD3 synaptic accumulation and MTOC translocation towards the antigen-presenting cell during immune synapsis. Remarkably, ISG20L2 controls the expression of immunoregulatory molecules, such as AHR, NKG2D, CTLA-4, CD137, TIM-3, PD-L1 or PD-1, which show increased levels in ISG20L2 knockout T cells. The dysregulation observed in these key molecules for T cell responses support a role for this exonuclease as a novel RNA-based regulator of T cell function.
- MeSH
- aktivace lymfocytů * MeSH
- antigen prezentující buňky MeSH
- endonukleasy MeSH
- lidé MeSH
- mikro RNA * genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
