DNA Topoisomerase I differentially modulates R-loops across the human genome

Manzo, Stefano Giustino; Hartono, Stella R.; Sanz, Lionel A.; Biasi, Sara De; Cossarizza, Andrea; Capranico, Giovanni; Chédin, Frédéric

doi:10.1101/257303

Cited by 14 publications

(23 citation statements)

References 80 publications

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“…During transcription TOP1 protein removes both negative and positive supercoils induced by RNA Pol progression. TOP1 plays a critical role in suppressing genome instability mediated by non-canonical secondary structures such as G4 and R-loops that are promoted by transcription (21)(22)(23)(24)(25). In humans, TOP2 activity is supported by two isoenzymes TOP2 alpha (TOP2A) and TOP2 beta (TOP2B), that are encoded by two different genes.…”

Section: Introductionmentioning

confidence: 99%

Transcription-associated topoisomerase activities control DNA-breaks production by G-quadruplex ligands

Pipier

Bossaert

Riou

et al. 2020

Preprint

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G-quadruplexes (G4), non-canonical DNA structures, are involved in several essential processes. Stabilization of G4 structures by small compounds (G4 ligands) affects almost all DNA transactions, including telomere maintenance and genomic stability. Here, thanks to a powerful and unbiased genetic approach, we identify topoisomerase 2-alpha (TOP2A) as the main effector of cell cytotoxicity induced by CX5461, a G4 ligand currently undergoing phase I/II clinical trials. This approach also allowed to identify new point mutations affecting TOP2A activity without compromising cell viability. Moreover, based on cross-resistance studies and siRNA-based protein depletion we report that TOP2A plays a major role in cell cytotoxicity induced by two unrelated clastogenic G4 ligands, CX5461 and pyridostatin (PDS). We also report that cytotoxic effects induced by both compounds are associated with topoisomerase 2mediated DNA breaks production. Finally, we show that TOP2-mediated DNA breaks production is strongly associated with RNA Pol II-dependent transcription and is countered by topoisomerase 1 (TOP1). Altogether our results indicate that clastogenic G4 ligands act as DNA structure-driven TOP2-poisons at transcribed regions bearing G-quadruplex structures.

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Section: Introductionmentioning

confidence: 99%

Transcription-associated topoisomerase activities control DNA-breaks production by G-quadruplex ligands

Pipier

Bossaert

Riou

et al. 2020

Preprint

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“…rDNA gene arrays represent the most abundantly transcribed regions of the human genome and have historically been considered a prominent source of R-loops in E. coli , yeast, and human cells [28-30]. To date, however, RNA Polymerase I-driven R-loops over rRNA genes have never been characterized at the single-molecule level.…”

Section: Resultsmentioning

confidence: 99%

High-Throughput Single-Molecule R-loop Footprinting Reveals Principles of R-loop Formation

Malig

Hartono²,

Giafaglione

et al. 2019

Preprint

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R-loops are a prevalent class of non-B DNA structures that form during transcription upon reannealing of the nascent RNA to the template DNA strand. R-loops have been profiled using the S9.6 antibody to immunoprecipitate DNA:RNA hybrids. S9.6-based DNA:RNA immunoprecipitation (DRIP) techniques revealed that R-loops form dynamically over conserved genic hotspots. We developed an orthogonal profiling methodology that queries R-loops via the presence of long stretches of single-stranded DNA on the looped-out strand. Non-denaturing sodium bisulfite treatment catalyzes the conversion of unpaired cytosines to uracils, creating permanent genetic tags for the position of an R-loop. Long read, single-molecule PacBio sequencing allows the identification of R-loop 'footprints' at near nucleotide resolution in a strand-specific manner on single DNA molecules and at ultra-deep coverage. Single-molecule R-loop footprinting (SMRF-seq) revealed a strong agreement between S9.6-and bisulfite-based R-loop mapping and confirmed that R-loops form from unspliced transcripts over genic hotspots. Using the largest single-molecule R-loop dataset to date, we show that individual R-loops generate overlapping sets of molecular clusters that pile-up through larger R-loop-prone zones. SMRF-seq further established that R-loop distribution patterns are driven by both intrinsic DNA sequence features and DNA topological constraints, revealing the principles of R-loop formation. KEYWORDSR-loops, DNA:RNA immunoprecipitation, SMRT sequencing, non-denaturing bisulfite conversion, DNA topology, S9.6 antibodydata analysis steps. Here we present a novel adaption of R-loop footprinting that permits singlemolecule R-loop detection at near nucleotide resolution in a strand-specific manner on long amplicons and at ultra-high coverage. This method and its accompanying computational analysis and data visualization pipeline enables deep, cost-effective, R-loop profiling at a range of genomic loci, under any condition and in any genome. SMRTbell library constructionWe used the PacBio RSII system to achieve long-read, single-molecule resolution sequencing of R-loop footprints. We generated libraries by pooling non-overlapping amplicons (less than 20 products per run) adding equal amounts for each. Starting with 1-2 µg of PCR products, pooled samples were concentrated using 1X Ampure bead wash. Libraries were built following the "Procedure & Checklist -2 kb Template Preparation and Sequencing" protocol (PN 001-143-835-08) from PacBio with a few modifications. No prior DNA damage repair step was done. AMPure bead wash steps were done using 0.8X concentration. Ligation was done for 1 hour at 25°C. SMRTbell libraries were quantified and size confirmation done by either by gel electrophoresis or Agilent Genomic's 2100 Bioanalyzer. Libraries were sequenced on a PacBio RSII instrument with 6-hour movie times. R-loop Profiling using DRIPc-seq.DRIPc-seq was applied to NTERA-2 cells as described [3] except a Ribonuclease A pretreatment was applied to the extracted nucleic...

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“…We found additional proteins described to bind to R-loops (99) and prevent their formation (100) in the BioID dataset. These include members of the spliceosome (101,102), the exosome (103), topoisomerase 1 (104) and an associated bromodomain-containing protein BRD2 (105), transcription elongation factor TFIIS (106) or FACT (107). These proteins are involved in the resolution of genome maintenance conflicts caused by R-loop formation in mammals.…”

Section: Discussionmentioning

confidence: 99%

A DOT1B/Ribonuclease H2 protein complex is involved in R-loop processing, genomic integrity and antigenic variation inTrypanosoma brucei

Eisenhuth

Vellmer

Butter

et al. 2020

Preprint

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The parasite Trypanosoma brucei periodically changes the expression of protective variant surface glycoproteins (VSGs) to evade its host's immune system in a process known as antigenic variation.One route to change VSG expression is the transcriptional activation of a previously silent VSG expression site (ES), a subtelomeric region containing the VSG genes. Homologous recombination of a different VSG from a large reservoir into the active ES represents another route. The conserved histone methyltransferase DOT1B is involved in transcriptional silencing of inactive ES and influences ES switching kinetics. The molecular machinery that enables DOT1B to execute these regulatory functions remains elusive, however. To better understand DOT1B-mediated regulatory processes, we purified DOT1B-associated proteins using complementary biochemical approaches. We identified several novel DOT1B-interactors. One of these was the Ribonuclease H2 complex, previously shown to resolve RNA-DNA hybrids, maintain genome integrity, and play a role in antigenic variation. Our study revealed that DOT1B depletion results in an increase in RNA-DNA hybrids, accumulation of DNA damage and recombination-based ES switching events. Surprisingly, a similar pattern of VSG deregulation was observed in Ribonuclease H2 mutants. We propose that both proteins act together in resolving R-loops to ensure genome integrity and contribute to the tightly-regulated process of antigenic variation.

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DNA Topoisomerase I differentially modulates R-loops across the human genome

Cited by 14 publications

References 80 publications

Transcription-associated topoisomerase activities control DNA-breaks production by G-quadruplex ligands

Transcription-associated topoisomerase activities control DNA-breaks production by G-quadruplex ligands

High-Throughput Single-Molecule R-loop Footprinting Reveals Principles of R-loop Formation

A DOT1B/Ribonuclease H2 protein complex is involved in R-loop processing, genomic integrity and antigenic variation inTrypanosoma brucei

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