DNA damage and genome instability by G-quadruplex ligands are mediated by R-loops in human cancer cells
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PROVIDER: PRJNA476585 | ENA |
REPOSITORIES: ENA
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Project description:G-quadruplexes (G4s) and R-loops are non-canonical DNA structures that can play regulatory functions of basic nuclear processes and can trigger DNA damage and genome instability. We here show that specific G4 ligands can stabilize G4s and simultaneously increase R-loop levels in human cancer cells likely by spreading DNA:RNA heteroduplexes to adjacent regions containing G4 structures. DNA cleavage and DNA damage response induced by G4 ligands were rescued by overexpression of exogenous RNaseH1 in cancer cells independently of BRCA2 status. The data thus show that R-loops are involved in the induction of DNA damage by chemical G4 stabilization. In addition, G4 ligands trigger the formation of micronuclei, particularly in BRCA2-silenced cancer cells, in an R-loop dependent manner. Our results uncover the mechanism of genome instability caused by G4 ligands and can open to the development of unexpected anticancer strategies using G4-targeted agents.
2018-12-27 | GSE115957 | GEO
Project description:VID22 counteracts G-quadruplex-induced genome instability
| PRJNA731119 | ENA
Project description:Genome instability is a condition characterized by the accumulation of various genetic alterations and is a hallmark of cancer cells. To uncover new genes and cellular pathways controlling the levels of endogenous DNA damage and affecting genome integrity, we exploited a Synthetic Genetic Array (SGA)-based screen in yeast. Among the positive genes, we identified VID22, a gene previously reported to be involved in DNA double-strand break repair. We found that vid22Δ cells exhibit chronic DNA damage response activation, show increased levels of increased endogenous DNA damage and accumulate DNA aberrations in sequences displaying high probabilities of forming G-quadruplexes (G4-DNA). If not resolved, these non-canonical DNA secondary structures can block the progression of both DNA and RNA polymerases and correlate with chromosome fragile sites. We show that Vid22 in vitro and in vivo binds directly to and protects DNA at G4-containing regions both in vitro and in vivo. In particular, loss of VID22 causes an increase in gross chromosomal rearrangement (GCR) events dependent on G-quadruplex forming sequences. We also demonstrate that the absence of Vid22 causes defects in the correct maintenance of G4-DNA rich elements, such as telomeres and mtDNA, and hypersensitivity to the G4-stabilizing ligand TMPyP4. We thus propose that Vid22 is directly involved in genome integrity maintenance as a novel regulator of G4 metabolism.
2021-11-08 | GSE174688 | GEO
Project description:G quadruplexes (G4s) and R loops are noncanonical DNA structures that can regulate basic nuclear processes and trigger DNA damage, genome instability, and cell killing. By different technical approaches, we here establish that specific G4 ligands stabilize G4s and simultaneously increase R-loop levels within minutes in human cancer cells. Genome-wide mapping of R loops showed that the studied G4 ligands likely cause the spreading of R loops to adjacent regions containing G4 structures, preferentially at 3'-end regions of expressed genes, which are partially ligand-specific. Overexpression of an exogenous human RNaseH1 rescued DNA damage induced by G4 ligands in BRCA2-proficient and BRCA2-silenced cancer cells. Moreover, even if the studied G4 ligands increased noncanonical DNA structures at similar levels in nuclear chromatin, their cellular effects were different in relation to cell-killing activity and stimulation of micronuclei, a hallmark of genome instability. Our findings therefore establish that G4 ligands can induce DNA damage by an R loop-dependent mechanism that can eventually lead to different cellular consequences depending on the chemical nature of the ligands.
| S-EPMC6338839 | biostudies-literature
Project description:The G-quadruplex is an alternative DNA structural motif that is considered to be functionally important in the mammalian genome. Herein, we address the hypothesis that G-quadruplex structures can exist within double-stranded genomic DNA using a G-quadruplex-specific probe. An engineered antibody is employed to enrich for DNA containing G-quadruplex structures, followed by deep sequencing to detect and map G-quadruplexes at high resolution in genomic DNA from human breast adenocarcinoma cells. Our high sensitivity structure-based pull-down strategy enables the isolation of genomic DNA fragments bearing a single as well as multiple G-quadruplex structures. Stable G-quadruplex structures are found in sub-telomeres, gene bodies and gene regulatory regions. For a sample of identified target genes, we show that G-quadruplex stabilizing ligands can modulate transcription. These results confirm the existence of G-quadruplex structures and their persistence in human genomic DNA. Four independent libraries have been enriched in DNA G-quadruplex structures using a G-quadruplex-specific probe. One genomic input library was sequenced as control. Deep-sequencing of these libraries allowed the mapping of G-quadruplexes on the genome.
2013-05-08 | E-GEOD-45241 | biostudies-arrayexpress
Project description:R loops are an important source of genome instability largely due to its negative impact on replication progression. Yra1/ALY is an abundant RNA-binding factor conserved from yeast to humans and required for mRNA export, but its excess cause lethality and genome instability. Here, we show that Yra1 binds RNA-DNA hybrids in vitro and when artificially overexpressed is recruited to chromatin in an RNA-DNA hybrid-dependent manner stabilizing R loops and converting them into replication obstacles in vivo. Importantly, excess of Yra1 increases R loop-mediated genome instability caused by transcription-replication collisions regardless of whether they are co-directional or head-on. It also induces telomere shortening and senescence, consistent with a defect in telomere replication. Our results indicate that R loops form transiently in cells regardless of replication and, after stabilization by excess Yra1, they compromise genome integrity, in agreement with a two-step model of R loop-mediated genome instability. This work opens new perspectives to understand transcription-associated genome instability in repair-deficient cells, including tumoral cells.
2018-06-27 | GSE113580 | GEO
Project description:Genome-wide Map of R-Loop-Induced Damage Reveals How a Subset of R-Loops Contributes to Genomic Instability
| PRJNA434004 | ENA
Project description:The G-quadruplex is an alternative DNA structural motif that is considered to be functionally important in the mammalian genome. Herein, we address the hypothesis that G-quadruplex structures can exist within double-stranded genomic DNA using a G-quadruplex-specific probe. An engineered antibody is employed to enrich for DNA containing G-quadruplex structures, followed by deep sequencing to detect and map G-quadruplexes at high resolution in genomic DNA from human breast adenocarcinoma cells. Our high sensitivity structure-based pull-down strategy enables the isolation of genomic DNA fragments bearing a single as well as multiple G-quadruplex structures. Stable G-quadruplex structures are found in sub-telomeres, gene bodies and gene regulatory regions. For a sample of identified target genes, we show that G-quadruplex stabilizing ligands can modulate transcription. These results confirm the existence of G-quadruplex structures and their persistence in human genomic DNA.
2013-05-08 | GSE45241 | GEO
Project description:Targeting G-quadruplex structures is currently viewed as a promising anticancer strategy. Searching for potent and selective G-quadruplex binders, here we describe a small series of new monohydrazone derivatives designed as analogues of a lead which was proved to stabilize G-quadruplex structures and increase R loop levels in human cancer cells. To investigate the G-quadruplex binding properties of the new molecules, in vitro biophysical studies were performed employing both telomeric and oncogene promoter G-quadruplex-forming sequences. The obtained results allowed the identification of a highly selective G-quadruplex ligand that, when studied in human cancer cells, proved to be able to stabilize both G-quadruplexes and R loops and showed a potent cell killing activity associated with the formation of micronuclei, a clear sign of genome instability.
| S-EPMC7997572 | biostudies-literature
Project description:A375 and HT1080 cells are treated with G-quadruplex ligands PDS or PhenDC3 following genome-wide shRNA knockdown. This enables the identification of genes that when silenced, specifically compromises cell growth in the presence of the ligand. First, a pilot screen was performed to determine a ligand concentration and experimental duration that caused ligand-specific, significant changes in shRNA levels. Second, a genome-wide screen was performed to globally evaluate G4-ligand synthetic lethal interactions. Third, to corroborate the G4-sensitisers uncovered in the genome-wide screen, a focussed screen was performed with a custom shRNA pool.
2019-06-22 | E-MTAB-6367 | biostudies-arrayexpress