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

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-helikasy * genetika   metabolismus   MeSH
• genetická transkripce MeSH
• lidé MeSH
• multifunkční enzymy genetika   metabolismus   MeSH
• neurodegenerativní nemoci MeSH
• R-smyčka MeSH
• regulace genové exprese MeSH
• RNA-helikasy * metabolismus   MeSH
• Saccharomyces cerevisiae - proteiny metabolismus   MeSH
• Saccharomyces cerevisiae metabolismus   MeSH
• terminace genetické transkripce * MeSH
• transkripční faktory genetika   metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH