RESUMO
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.
Assuntos
Sítios Frágeis do Cromossomo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/biossíntese , Endonucleases/metabolismo , Mitose , RecQ Helicases/metabolismo , Origem de Replicação , Sítios de Ligação , Proteína Quinase CDC2 , Instabilidade Cromossômica , Segregação de Cromossomos , Quinases Ciclina-Dependentes/metabolismo , DNA/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Células HEK293 , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Interferência de RNA , Rad51 Recombinase/metabolismo , RecQ Helicases/genética , Fatores de Tempo , TransfecçãoRESUMO
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.
Assuntos
DNA Helicases , RNA Helicases , Terminação da Transcrição Genética , Humanos , DNA Helicases/genética , DNA Helicases/metabolismo , Regulação da Expressão Gênica , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Doenças Neurodegenerativas , Estruturas R-Loop , RNA Helicases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
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.
Assuntos
Replicação do DNA , Estruturas R-Loop , Humanos , Replicação do DNA/genética , DNA Helicases/metabolismo , Endonucleases/metabolismo , DNA/metabolismo , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismoRESUMO
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.