RECQ5 belongs to the RecQ family of DNA helicases. It is conserved from Drosophila to humans and its deficiency results in genomic instability and cancer susceptibility in mice. Human RECQ5 is known for its ability to regulate homologous recombination by disrupting RAD51 nucleoprotein filaments. It also binds to RNA polymerase II (RNAPII) and negatively regulates transcript elongation by RNAPII. Here, we summarize recent studies implicating RECQ5 in the prevention and resolution of transcription-replication conflicts, a major intrinsic source of genomic instability during cancer development.

• MeSH
• DNA genetika   metabolismus   MeSH
• genetická transkripce genetika   MeSH
• helikasy RecQ genetika   metabolismus   fyziologie   MeSH
• lidé MeSH
• nestabilita genomu MeSH
• replikace DNA MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Collisions between replication and transcription machineries represent a significant source of genomic instability. RECQ5 DNA helicase binds to RNA-polymerase (RNAP) II during transcription elongation and suppresses transcription-associated genomic instability. Here, we show that RECQ5 also associates with RNAPI and enforces the stability of ribosomal DNA arrays. We demonstrate that RECQ5 associates with transcription complexes in DNA replication foci and counteracts replication fork stalling in RNAPI- and RNAPII-transcribed genes, suggesting that RECQ5 exerts its genome-stabilizing effect by acting at sites of replication-transcription collisions. Moreover, RECQ5-deficient cells accumulate RAD18 foci and BRCA1-dependent RAD51 foci that are both formed at sites of interference between replication and transcription and likely represent unresolved replication intermediates. Finally, we provide evidence for a novel mechanism of resolution of replication-transcription collisions wherein the interaction between RECQ5 and proliferating cell nuclear antigen (PCNA) promotes RAD18-dependent PCNA ubiquitination and the helicase activity of RECQ5 promotes the processing of replication intermediates.

• MeSH
• biologické modely MeSH
• DNA řízené RNA-polymerasy metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-dependentní DNA-polymerasy metabolismus   MeSH
• elongace genetické transkripce MeSH
• fyziologický stres genetika   MeSH
• genetická transkripce * MeSH
• HEK293 buňky MeSH
• helikasy RecQ metabolismus   MeSH
• interakční proteinové domény a motivy MeSH
• lidé MeSH
• multienzymové komplexy metabolismus   MeSH
• otevřené čtecí rámce genetika   MeSH
• prekurzory RNA genetika   MeSH
• proliferační antigen buněčného jádra metabolismus   MeSH
• protein BRCA1 metabolismus   MeSH
• rekombinasa Rad51 metabolismus   MeSH
• replikace DNA * MeSH
• ribozomální DNA metabolismus   MeSH
• ubikvitinace MeSH
• ubikvitinligasy metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

The SLX4 tumor suppressor is a scaffold that plays a pivotal role in several aspects of genome protection, including homologous recombination, interstrand DNA crosslink repair and the maintenance of common fragile sites and telomeres. Here, we unravel an unexpected direct interaction between SLX4 and the DNA helicase RTEL1, which, until now, were viewed as having independent and antagonistic functions. We identify cancer and Hoyeraal-Hreidarsson syndrome-associated mutations in SLX4 and RTEL1, respectively, that abolish SLX4-RTEL1 complex formation. We show that both proteins are recruited to nascent DNA, tightly co-localize with active RNA pol II, and that SLX4, in complex with RTEL1, promotes FANCD2/RNA pol II co-localization. Importantly, disrupting the SLX4-RTEL1 interaction leads to DNA replication defects in unstressed cells, which are rescued by inhibiting transcription. Our data demonstrate that SLX4 and RTEL1 interact to prevent replication-transcription conflicts and provide evidence that this is independent of the nuclease scaffold function of SLX4.

• MeSH
• DNA-helikasy genetika   metabolismus   MeSH
• dyskeratosis congenita genetika   MeSH
• genetická transkripce * MeSH
• HeLa buňky MeSH
• lidé MeSH
• mentální retardace genetika   MeSH
• mikrocefalie genetika   MeSH
• protein FANCD2 genetika   metabolismus   MeSH
• rekombinasy genetika   metabolismus   MeSH
• replikace DNA * MeSH
• růstová retardace plodu genetika   MeSH
• zárodečné mutace MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• MeSH
• DNA opravný a rekombinační protein Rad52 metabolismus   MeSH
• DNA vazebné proteiny metabolismus   MeSH
• DNA-ligasy metabolismus   MeSH
• DNA-polymerasa III metabolismus   MeSH
• endodeoxyribonukleasy metabolismus   MeSH
• endonukleasy genetika   metabolismus   MeSH
• genetická transkripce genetika   MeSH
• HeLa buňky MeSH
• helikasy RecQ metabolismus   fyziologie   MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• R-smyčka genetika   fyziologie   MeSH
• rekombinasa Rad51 genetika   metabolismus   fyziologie   MeSH
• replikace DNA genetika   fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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

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

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

STUDY HYPOTHESIS: In women with preterm premature rupture of the membranes (PPROM), increased oxidative stress may accelerate premature cellular senescence, senescence-associated inflammation and proteolysis, which may predispose them to rupture. STUDY FINDING: We demonstrate mechanistic differences between preterm birth (PTB) and PPROM by revealing differences in fetal membrane redox status, oxidative stress-induced damage, distinct signaling pathways and senescence activation. WHAT IS KNOWN ALREADY: Oxidative stress-associated fetal membrane damage and cell cycle arrest determine adverse pregnancy outcomes, such as spontaneous PTB and PPROM. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: Fetal membranes and amniotic fluid samples were collected from women with PTB and PPROM. Molecular, biochemical and histologic markers were used to document differences in oxidative stress and antioxidant enzyme status, DNA damage, secondary signaling activation by Ras-GTPase and mitogen-activated protein kinases, and activation of senescence between membranes from the two groups. MAIN RESULTS AND THE ROLE OF CHANCE: Oxidative stress was higher and antioxidant enzymes were lower in PPROM compared with PTB. PTB membranes had minimal DNA damage and showed activation of Ras-GTPase and ERK/JNK signaling pathway with minimal signs of senescence. PPROM had higher numbers of cells with DNA damage, prosenescence stress kinase (p38 MAPK) activation and signs of senescence. LIMITATIONS, REASONS FOR CAUTION: Samples were obtained retrospectively after delivery. The markers of senescence that we tested are specific but are not sufficient to confirm senescence as the pathology in PPROM. WIDER IMPLICATIONS OF THE FINDINGS: Oxidative stress-induced DNA damage and senescence are characteristics of fetal membranes from PPROM, compared with PTB with intact membranes. PTB and PPROM arise from distinct pathophysiologic pathways. Oxidative stress and oxidative stress-induced cellular damages are likely determinants of the mechanistic signaling pathways and phenotypic outcome. STUDY FUNDING AND COMPETING INTERESTS: This study is supported by developmental funds to Dr R. Menon from the Department of Obstetrics and Gynecology at The University of Texas Medical Branch at Galveston and funds to Dr M. Kacerovský from the Ministry of Health Czech Republic (UHHK, 001799906). The authors report no conflict of interest.

• MeSH
• dospělí MeSH
• extraembryonální obaly zranění   metabolismus   MeSH
• homeodoménové proteiny genetika   metabolismus   MeSH
• lamin typ B genetika   metabolismus   MeSH
• lidé MeSH
• MAP kinasa-kinasa 4 genetika   metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 1 genetika   metabolismus   MeSH
• mitogenem aktivovaná proteinkinasa 3 genetika   metabolismus   MeSH
• mitogenem aktivované proteinkinasy p38 genetika   metabolismus   MeSH
• novorozenec nedonošený MeSH
• novorozenec MeSH
• oxidační stres genetika   MeSH
• poškození DNA MeSH
• předčasný odtok plodové vody genetika   metabolismus   patologie   MeSH
• předčasný porod MeSH
• regulace genové exprese MeSH
• signální transdukce genetika   MeSH
• stanovení celkové genové exprese MeSH
• stárnutí buněk MeSH
• superoxiddismutasa genetika   metabolismus   MeSH
• těhotenství MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• novorozenec MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH