RESUMO
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.
Assuntos
RNA Helicases DEAD-box , DNA Helicases , Replicação do DNA , Proteínas de Ligação a DNA , Endonucleases , Enzimas Multifuncionais , Estruturas R-Loop , RNA Helicases , DNA Helicases/metabolismo , DNA Helicases/genética , RNA Helicases/metabolismo , RNA Helicases/genética , Humanos , Enzimas Multifuncionais/metabolismo , Enzimas Multifuncionais/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , RNA Helicases DEAD-box/metabolismo , RNA Helicases DEAD-box/genética , Endonucleases/metabolismo , Endonucleases/genética , Endonucleases Flap/metabolismo , Endonucleases Flap/genética , Transcrição Gênica , DNA Ligase Dependente de ATP/metabolismo , DNA Ligase Dependente de ATP/genética , DNA/metabolismo , DNA/genéticaRESUMO
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 MutSß, an MLH1-PMS1 heterodimer termed MutLß, 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 MutSß, the helicase activity of FANCJ, and the binding of FANCJ to MLH1. Furthermore, we show that MutSß, MutLß, and MLH1-FANCJ interaction mediate FANCJ recruitment to G4s. These data suggest that MutSß, MutLß, and FANCJ act in conjunction to eliminate G4/R-loops at TRC sites, allowing replication restart.
Assuntos
Proteínas de Grupos de Complementação da Anemia de Fanconi , Estruturas R-Loop , Humanos , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA/genéticaRESUMO
Elevated levels of reactive oxygen species (ROS) reduce replication fork velocity by causing dissociation of the TIMELESS-TIPIN complex from the replisome. Here, we show that ROS generated by exposure of human cells to the ribonucleotide reductase inhibitor hydroxyurea (HU) promote replication fork reversal in a manner dependent on active transcription and formation of co-transcriptional RNA:DNA hybrids (R-loops). The frequency of R-loop-dependent fork stalling events is also increased after TIMELESS depletion or a partial inhibition of replicative DNA polymerases by aphidicolin, suggesting that this phenomenon is due to a global replication slowdown. In contrast, replication arrest caused by HU-induced depletion of deoxynucleotides does not induce fork reversal but, if allowed to persist, leads to extensive R-loop-independent DNA breakage during S-phase. Our work reveals a link between oxidative stress and transcription-replication interference that causes genomic alterations recurrently found in human cancer.