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

A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81-Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81-Mms4. In this study, we show that the Srs2 and Mus81-Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81-Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81-Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA. Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.

• MeSH
• "flap" endonukleasy fyziologie   MeSH
• DNA primery MeSH
• DNA vazebné proteiny fyziologie   MeSH
• DNA-helikasy fyziologie   MeSH
• endonukleasy fyziologie   MeSH
• fluorescenční mikroskopie MeSH
• polymerázová řetězová reakce MeSH
• rekombinace genetická * MeSH
• Saccharomyces cerevisiae - proteiny fyziologie   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• sekvence nukleotidů MeSH
• techniky dvojhybridového systému MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH

BACKGROUND: Proper DNA replication is essential for faithful transmission of the genome. However, replication stress has serious impact on the integrity of the cell, leading to stalling or collapse of replication forks, and has been determined as a driving force of carcinogenesis. Mus81-Mms4 complex is a structure-specific endonuclease previously shown to be involved in processing of aberrant replication intermediates and promotes POLD3-dependent DNA synthesis via break-induced replication. However, how replication components might be involved in this process is not known. RESULTS: Herein, we show the interaction and robust stimulation of Mus81-Mms4 nuclease activity by heteropentameric replication factor C (RFC) complex, the processivity factor of replicative DNA polymerases that is responsible for loading of proliferating cell nuclear antigen (PCNA) during DNA replication and repair. This stimulation is enhanced by RFC-dependent ATP hydrolysis and by PCNA loading on the DNA. Moreover, this stimulation is not specific to Rfc1, the largest of subunit of this complex, thus indicating that alternative clamp loaders may also play a role in the stimulation. We also observed a targeting of Mus81 by RFC to the nick-containing DNA substrate and we provide further evidence that indicates cooperation between Mus81 and the RFC complex in the repair of DNA lesions generated by various DNA-damaging agents. CONCLUSIONS: Identification of new interacting partners and modulators of Mus81-Mms4 nuclease, RFC, and PCNA imply the cooperation of these factors in resolution of stalled replication forks and branched DNA structures emanating from the restarted replication forks under conditions of replication stress.

• MeSH
• "flap" endonukleasy genetika   metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• endonukleasy genetika   metabolismus   MeSH
• proliferační antigen buněčného jádra genetika   metabolismus   MeSH
• rekombinace genetická MeSH
• replikace DNA MeSH
• replikační protein C genetika   metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

BACKGROUND: DNA-protein cross-links (DPCs) are one of the most deleterious DNA lesions, originating from various sources, including enzymatic activity. For instance, topoisomerases, which play a fundamental role in DNA metabolic processes such as replication and transcription, can be trapped and remain covalently bound to DNA in the presence of poisons or nearby DNA damage. Given the complexity of individual DPCs, numerous repair pathways have been described. The protein tyrosyl-DNA phosphodiesterase 1 (Tdp1) has been demonstrated to be responsible for removing topoisomerase 1 (Top1). Nevertheless, studies in budding yeast have indicated that alternative pathways involving Mus81, a structure-specific DNA endonuclease, could also remove Top1 and other DPCs. RESULTS: This study shows that MUS81 can efficiently cleave various DNA substrates modified by fluorescein, streptavidin or proteolytically processed topoisomerase. Furthermore, the inability of MUS81 to cleave substrates bearing native TOP1 suggests that TOP1 must be either dislodged or partially degraded prior to MUS81 cleavage. We demonstrated that MUS81 could cleave a model DPC in nuclear extracts and that depletion of TDP1 in MUS81-KO cells induces sensitivity to the TOP1 poison camptothecin (CPT) and affects cell proliferation. This sensitivity is only partially suppressed by TOP1 depletion, indicating that other DPCs might require the MUS81 activity for cell proliferation. CONCLUSIONS: Our data indicate that MUS81 and TDP1 play independent roles in the repair of CPT-induced lesions, thus representing new therapeutic targets for cancer cell sensitisation in combination with TOP1 inhibitors.

• MeSH
• DNA vazebné proteiny * genetika   metabolismus   MeSH
• DNA-topoisomerasy I genetika   metabolismus   MeSH
• endonukleasy * genetika   metabolismus   MeSH
• fosfodiesterasy * genetika   metabolismus   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• Saccharomyces cerevisiae - proteiny * genetika   metabolismus   MeSH
• Saccharomyces cerevisiae MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Replication across damaged DNA templates is accompanied by transient formation of sister chromatid junctions (SCJs). Cells lacking Esc2, an adaptor protein containing no known enzymatic domains, are defective in the metabolism of these SCJs. However, how Esc2 is involved in the metabolism of SCJs remains elusive. Here we show interaction between Esc2 and a structure-specific endonuclease Mus81-Mms4 (the Mus81 complex), their involvement in the metabolism of SCJs, and the effects Esc2 has on the enzymatic activity of the Mus81 complex. We found that Esc2 specifically interacts with the Mus81 complex via its SUMO-like domains, stimulates enzymatic activity of the Mus81 complex in vitro, and is involved in the Mus81 complex-dependent resolution of SCJs in vivo Collectively, our data point to the possibility that the involvement of Esc2 in the metabolism of SCJs is, in part, via modulation of the activity of the Mus81 complex.

• MeSH
• chromatidy chemie   metabolismus   MeSH
• DNA fungální genetika   metabolismus   MeSH
• DNA vazebné proteiny chemie   genetika   metabolismus   MeSH
• endonukleasy chemie   genetika   metabolismus   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• jaderné proteiny chemie   genetika   metabolismus   MeSH
• klonování DNA MeSH
• křížová struktura DNA chemie   metabolismus   MeSH
• malé modifikační proteiny související s ubikvitinem chemie   genetika   metabolismus   MeSH
• nestabilita genomu MeSH
• poškození DNA MeSH
• proteinové domény MeSH
• regulace genové exprese u hub * MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• replikace DNA MeSH
• Saccharomyces cerevisiae - proteiny chemie   genetika   metabolismus   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• vazba proteinů 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 spojení metabolismus   genetika   MeSH
• Saccharomyces cerevisiae - proteiny * metabolismus   genetika   MeSH
• Saccharomyces cerevisiae genetika   metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.

• MeSH
• časové faktory MeSH
• chromozomální nestabilita MeSH
• cyklin-dependentní kinasy metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• DNA biosyntéza   genetika   MeSH
• endodeoxyribonukleasy metabolismus   MeSH
• endonukleasy genetika   metabolismus   MeSH
• fosforylace MeSH
• fragilní místa na chromozomu * MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• helikasy RecQ genetika   metabolismus   MeSH
• lidé MeSH
• mitóza * MeSH
• oprava DNA * MeSH
• poškození DNA MeSH
• rekombinasa Rad51 metabolismus   MeSH
• replikační počátek * MeSH
• RNA interference MeSH
• segregace chromozomů MeSH
• transfekce MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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" 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

• Publikační typ
• abstrakty MeSH

• Publikační typ
• abstrakt z konference MeSH