R loops are nucleic acid structures comprising an DNA–RNA hybrid and a displaced single-stranded DNA. These structures may occur transiently during transcription, playing essential biological ...functions. However, persistent R loops may become pathological as they are important drivers of genome instability and have been associated with human diseases. The mitochondrial degradosome is a functionally conserved complex from bacteria to human mitochondria. It is composed of the ATP-dependent RNA and DNA helicase SUV3 and the PNPase ribonuclease, playing a central role in mitochondrial RNA surveillance and degradation. Here we describe a new role for the mitochondrial degradosome in preventing the accumulation of pathological R loops in the mitochondrial DNA, in addition to preventing dsRNA accumulation. Our data indicate that, similar to the molecular mechanisms acting in the nucleus, RNA surveillance mechanisms in the mitochondria are crucial to maintain its genome integrity by counteracting pathological R-loop accumulation.
The maintenance of genomic stability requires the coordination of multiple cellular tasks upon the appearance of DNA lesions. RNA editing, the post-transcriptional sequence alteration of RNA, has a ...profound effect on cell homeostasis, but its implication in the response to DNA damage was not previously explored. Here we show that, in response to DNA breaks, an overall change of the Adenosine-to-Inosine RNA editing is observed, a phenomenon we call the RNA Editing DAmage Response (REDAR). REDAR relies on the checkpoint kinase ATR and the recombination factor CtIP. Moreover, depletion of the RNA editing enzyme ADAR2 renders cells hypersensitive to genotoxic agents, increases genomic instability and hampers homologous recombination by impairing DNA resection. Such a role of ADAR2 in DNA repair goes beyond the recoding of specific transcripts, but depends on ADAR2 editing DNA:RNA hybrids to ease their dissolution.
Abstract
Although human nucleoporin Tpr is frequently deregulated in cancer, its roles are poorly understood. Here we show that Tpr depletion generates transcription-dependent replication stress, DNA ...breaks, and genomic instability. DNA fiber assays and electron microscopy visualization of replication intermediates show that Tpr deficient cells exhibit slow and asymmetric replication forks under replication stress. Tpr deficiency evokes enhanced levels of DNA-RNA hybrids. Additionally, complementary proteomic strategies identify a network of Tpr-interacting proteins mediating RNA processing, such as MATR3 and SUGP2, and functional experiments confirm that their depletion trigger cellular phenotypes shared with Tpr deficiency. Mechanistic studies reveal the interplay of Tpr with GANP, a component of the TREX-2 complex. The Tpr-GANP interaction is supported by their shared protein level alterations in a cohort of ovarian carcinomas. Our results reveal links between nucleoporins, DNA transcription and replication, and the existence of a network physically connecting replication forks with transcription, splicing, and mRNA export machinery.
Co-transcriptional RNA-DNA hybrids (R loops) cause genome instability. To prevent harmful R loop accumulation, cells have evolved specific eukaryotic factors, one being the BRCA2 double-strand break ...repair protein. As BRCA2 also protects stalled replication forks and is the FANCD1 member of the Fanconi Anemia (FA) pathway, we investigated the FA role in R loop-dependent genome instability. Using human and murine cells defective in FANCD2 or FANCA and primary bone marrow cells from FANCD2 deficient mice, we show that the FA pathway removes R loops, and that many DNA breaks accumulated in FA cells are R loop-dependent. Importantly, FANCD2 foci in untreated and MMC-treated cells are largely R loop dependent, suggesting that the FA functions at R loop-containing sites. We conclude that co-transcriptional R loops and R loop-mediated DNA damage greatly contribute to genome instability and that one major function of the FA pathway is to protect cells from R loops.
DNA-RNA hybrids play a physiological role in cellular processes, but often, they represent non-scheduled co-transcriptional structures with a negative impact on transcription, replication and DNA ...repair. Accumulating evidence suggests that they constitute a source of replication stress, DNA breaks and genome instability. Reciprocally, DNA breaks facilitate DNA-RNA hybrid formation by releasing the double helix torsional conformation. Cells avoid DNA-RNA accumulation by either preventing or removing hybrids directly or by DNA repair-coupled mechanisms. Given the R-loop impact on chromatin and genome organization and its potential relation with genetic diseases, we review R-loop homeostasis as well as their physiological and pathological roles.
DNA-RNA hybrids play a physiological role in cellular processes, but often, they represent non-scheduled co-transcriptional structures with a negative impact on transcription, replication and DNA repair. Accumulating evidence suggests that they constitute a source of replication stress, DNA breaks and genome instability. Reciprocally, DNA breaks facilitate DNA-RNA hybrid formation by releasing the double helix torsional conformation. Cells avoid DNA-RNA accumulation by either preventing or removing hybrids directly or by DNA repair-coupled mechanisms. Given the R-loop impact on chromatin and genome organization and its potential relation with genetic diseases, we review R-loop homeostasis as well as their physiological and pathological roles.
R loops are transcription byproducts that constitute a threat to genome integrity. Here we show that R loops are tightly linked to histone H3 S10 phosphorylation (H3S10P), a mark of chromatin ...condensation. Chromatin immunoprecipitation (ChIP)-on-chip (ChIP-chip) analyses reveal H3S10P accumulation at centromeres, pericentromeric chromatin, and a large number of active open reading frames (ORFs) in R-loop-accumulating yeast cells, better observed in G1. Histone H3S10 plays a key role in maintaining genome stability, as scored by ectopic recombination and plasmid loss, Rad52 foci, and Rad53 checkpoint activation. H3S10P coincides with the presence of DNA-RNA hybrids, is suppressed by ribonuclease H overexpression, and causes reduced accessibility of restriction endonucleases, implying a tight connection between R loops, H3S10P, and chromatin compaction. Such histone modifications were also observed in R-loop-accumulating Caenorhabditis elegans and HeLa cells. We therefore provide a role of RNA in chromatin structure essential to understand how R loops modulate genome dynamics.
Transcription is a central aspect of DNA metabolism that takes place on the same substrate as replication, repair and recombination Not surprisingly, therefore, there is a physical and functional ...connection between these processes In recent years, transcription has proven to be a relevant player in the maintenance of genome integrity and in the induction of genetic instability and diversity The aim of this review is to provide an integrative view on how transcription can control different aspects of genomic integrity, by exploring different mechanisms that might be responsible for transcription‐associated mutation (TAM) and transcription‐associated recombination (TAR).
Genetic instability, a phenomenon relevant for developmentally regulated processes, cancer, and inherited disorders, can be induced by transcription. However, the mechanisms of ...transcription-associated genetic instability are not yet understood. Analysis of
S. cerevisiae mutants of THO/TREX, a conserved eukaryotic protein complex functioning at the interface of transcription and mRNA metabolism, has provided evidence that transcription elongation impairment can cause hyperrecombination. Here we show, using
hpr1Δ mutants, that the nascent mRNA can diminish transcription elongation efficiency and promote recombination. If during transcription the nascent mRNA is self-cleaved by a hammerhead ribozyme, the transcription-defect and hyperrecombination phenotypes of
hpr1Δ cells are suppressed. Abolishment of hyperrecombination by overexpression of RNase H1 and molecular detection of DNA:RNA hybrids indicate that these are formed cotranscriptionally in
hpr1Δ cells. These data support a model to explain the connection between recombination, transcription, and mRNA metabolism and provide a new perspective to understanding transcription-associated recombination.
Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find ...that fork reversal and dormant origin firing due to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2, or inner-basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.
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► Gene gating inactivation suppresses fork collapse in checkpoint mutants ► The Mec1 checkpoint releases transcribed chromatin from nuclear pore complexes ► Mec1 phosphorylates Mlp1 nucleoporin to release topological blocks at gated genes ► Releasing local topology by double-strand break formation counteracts fork collapse
Abstract
BRCA1
or
BRCA2
germline mutations predispose to breast, ovarian and other cancers. High-throughput sequencing of tumour genomes revealed that oncogene amplification and
BRCA1/2
mutations are ...mutually exclusive in cancer, however the molecular mechanism underlying this incompatibility remains unknown. Here, we report that activation of β-catenin, an oncogene of the WNT signalling pathway, inhibits proliferation of BRCA1/2-deficient cells. RNA-seq analyses revealed β-catenin-induced discrete transcriptome alterations in BRCA2-deficient cells, including suppression of
CDKN1A
gene encoding the CDK inhibitor p21. This accelerates G1/S transition, triggering illegitimate origin firing and DNA damage. In addition, β-catenin activation accelerates replication fork progression in BRCA2-deficient cells, which is critically dependent on p21 downregulation. Importantly, we find that upregulated p21 expression is essential for the survival of BRCA2-deficient cells and tumours. Thus, our work demonstrates that β-catenin toxicity in cancer cells with compromised BRCA1/2 function is driven by transcriptional alterations that cause aberrant replication and inflict DNA damage.
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