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

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