21 research outputs found
DNA double-strand break formation and signalling in response to transcription-blocking topoisomerase I complexes
La topoisomĂ©rase I (Top1) Ă©limine les surenroulements de l'ADN gĂ©nĂ©rĂ©s lors de la transcription en produisant transitoirement des complexes de clivage Top1-ADN (Top1cc). Ces Top1cc transitoires peuvent ĂȘtre stabilisĂ©s par les camptothĂ©cines, dont sont dĂ©rivĂ©s des agents anticancĂ©reux, et par les frĂ©quentes altĂ©rations de l'ADN. Bien que les Top1cc stabilisĂ©s soient des lĂ©sions qui bloquent efficacement la transcription, la comprĂ©hension des processus molĂ©culaires qui rĂ©sultent du blocage des complexes transcriptionnels par les Top1cc est encore limitĂ©e. Des travaux prĂ©cĂ©dents ont montrĂ© que les Top1cc stabilisĂ©s produisent des cassures double-brin (DSBs) de l'ADN dĂ©pendantes de la transcription qui activent ATM. Dans ce projet, nous avons utilisĂ© des cellules quiescentes traitĂ©es avec la camptothĂ©cine pour induire des Top1cc bloquant la transcription et nous avons Ă©tudiĂ© les mĂ©canismes de la production et de la signalisation des DSBs. Nous montrons que les DSBs sont produites prĂ©fĂ©rentiellement dans les rĂ©gions sub-tĂ©lomĂ©riques lors de la rĂ©paration des Top1cc bloquant la transcription par les cassures simple-brin de l'ADN gĂ©nĂ©rĂ©es aprĂšs la protĂ©olyse de la Top1 et avant l'action de Tdp1. L'analyse de la signalisation de ces DSBs rĂ©vĂšle une nouvelle fonction de DNA-PK dans la promotion de l'ubiquitinylation conduisant (i) Ă l'activitĂ© complĂšte d'ATM aux sites des DSBs en favorisant l'ubiquitination d'H2AX et H2A, et (ii) Ă l'augmentation de la rĂ©paration des Top1cc en favorisant la protĂ©olyse de la Top1. Enfin, nous montrons que les DSBs co-transcriptionnelles induisent la mort des cellules quiescentes. L'ensemble de ces rĂ©sultats apportent un nouvel aperçu des rĂ©ponses cellulaires aux camptothĂ©cines, et suggĂšrent que les DSBs qui rĂ©sultent des Top1cc bloquant la transcription puissent contribuer Ă la pathogĂ©nĂšse du syndrome neurodĂ©gĂ©nĂ©ratif SCAN1, qui est causĂ© par une dĂ©ficience en Tdp1.Topoisomerase I (Top1) removes DNA supercoiling generated during transcription by producing Top1-DNA cleavage complexes (Top1cc). These transient Top1cc can be stabilized by camptothecins, from which anticancer drugs are derived, and by common DNA alterations. Although stabilized Top1cc are potent transcription-blocking lesions, our understanding regarding the molecular processes resulting from the stalling of transcription complexes by Top1cc is currently limited. Previous work showed that stabilized Top1cc produce transcription-dependent DNA double-strand breaks (DSBs) that activate ATM signalling. In this project, we used camptothecin-treated quiescent cells to induce transcription-blocking Top1cc and study the mechanisms of DSB production and signalling. We show that DSBs form preferentially at subtelomeric regions during the repair of transcription-blocking Top1cc from DNA single-strand breaks generated after Top1 proteolysis and before Tdp1 action. Analysis of DSB signalling reveals a novel function of DNA-PK in promoting protein ubiquitination leading (i) to full ATM activity at DSB sites by promoting H2AX and H2A ubiquitination, and (ii) to enhancement of Top1cc repair by promoting Top1 proteolysis. Finally, we show that co-transcriptional DSBs kill quiescent cells. Together, these findings provide new insights into the cellular responses to camptothecins and further suggest that DSBs arising from transcription-blocking Top1cc may contribute to the pathogenesis of the neurodegenerative SCAN1 syndrome, which is caused by Tdp1 deficiency
N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells
© 2019, The Author(s), under exclusive licence to Springer Nature America, Inc. R-loops are nucleic acid structures formed by an RNA:DNA hybrid and unpaired single-stranded DNA that represent a source of genomic instability in mammalian cells1â4. Here we show that N6-methyladenosine (m6A) modification, contributing to different aspects of messenger RNA metabolism5,6, is detectable on the majority of RNA:DNA hybrids in human pluripotent stem cells. We demonstrate that m6A-containing R-loops accumulate during G2/M and are depleted at G0/G1 phases of the cell cycle, and that the m6A reader promoting mRNA degradation, YTHDF2 (ref. 7), interacts with R-loop-enriched loci in dividing cells. Consequently, YTHDF2 knockout leads to increased R-loop levels, cell growth retardation and accumulation of ÎłH2AX, a marker for DNA double-strand breaks, in mammalian cells. Our results suggest that m6A regulates accumulation of R-loops, implying a role for this modification in safeguarding genomic stability
Formation et signalisation des cassures double-brin de l'ADN lors d'un blocage de la transcription
Topoisomerase I (Top1) removes DNA supercoiling generated during transcription by producing Top1-DNA cleavage complexes (Top1cc). These transient Top1cc can be stabilized by camptothecins, from which anticancer drugs are derived, and by common DNA alterations. Although stabilized Top1cc are potent transcription-blocking lesions, our understanding regarding the molecular processes resulting from the stalling of transcription complexes by Top1cc is currently limited. Previous work showed that stabilized Top1cc produce transcription-dependent DNA double-strand breaks (DSBs) that activate ATM signalling. In this project, we used camptothecin-treated quiescent cells to induce transcription-blocking Top1cc and study the mechanisms of DSB production and signalling. We show that DSBs form preferentially at subtelomeric regions during the repair of transcription-blocking Top1cc from DNA single-strand breaks generated after Top1 proteolysis and before Tdp1 action. Analysis of DSB signalling reveals a novel function of DNA-PK in promoting protein ubiquitination leading (i) to full ATM activity at DSB sites by promoting H2AX and H2A ubiquitination, and (ii) to enhancement of Top1cc repair by promoting Top1 proteolysis. Finally, we show that co-transcriptional DSBs kill quiescent cells. Together, these findings provide new insights into the cellular responses to camptothecins and further suggest that DSBs arising from transcription-blocking Top1cc may contribute to the pathogenesis of the neurodegenerative SCAN1 syndrome, which is caused by Tdp1 deficiency.La topoisomĂ©rase I (Top1) Ă©limine les surenroulements de l'ADN gĂ©nĂ©rĂ©s lors de la transcription en produisant transitoirement des complexes de clivage Top1-ADN (Top1cc). Ces Top1cc transitoires peuvent ĂȘtre stabilisĂ©s par les camptothĂ©cines, dont sont dĂ©rivĂ©s des agents anticancĂ©reux, et par les frĂ©quentes altĂ©rations de l'ADN. Bien que les Top1cc stabilisĂ©s soient des lĂ©sions qui bloquent efficacement la transcription, la comprĂ©hension des processus molĂ©culaires qui rĂ©sultent du blocage des complexes transcriptionnels par les Top1cc est encore limitĂ©e. Des travaux prĂ©cĂ©dents ont montrĂ© que les Top1cc stabilisĂ©s produisent des cassures double-brin (DSBs) de l'ADN dĂ©pendantes de la transcription qui activent ATM. Dans ce projet, nous avons utilisĂ© des cellules quiescentes traitĂ©es avec la camptothĂ©cine pour induire des Top1cc bloquant la transcription et nous avons Ă©tudiĂ© les mĂ©canismes de la production et de la signalisation des DSBs. Nous montrons que les DSBs sont produites prĂ©fĂ©rentiellement dans les rĂ©gions sub-tĂ©lomĂ©riques lors de la rĂ©paration des Top1cc bloquant la transcription par les cassures simple-brin de l'ADN gĂ©nĂ©rĂ©es aprĂšs la protĂ©olyse de la Top1 et avant l'action de Tdp1. L'analyse de la signalisation de ces DSBs rĂ©vĂšle une nouvelle fonction de DNA-PK dans la promotion de l'ubiquitinylation conduisant (i) Ă l'activitĂ© complĂšte d'ATM aux sites des DSBs en favorisant l'ubiquitination d'H2AX et H2A, et (ii) Ă l'augmentation de la rĂ©paration des Top1cc en favorisant la protĂ©olyse de la Top1. Enfin, nous montrons que les DSBs co-transcriptionnelles induisent la mort des cellules quiescentes. L'ensemble de ces rĂ©sultats apportent un nouvel aperçu des rĂ©ponses cellulaires aux camptothĂ©cines, et suggĂšrent que les DSBs qui rĂ©sultent des Top1cc bloquant la transcription puissent contribuer Ă la pathogĂ©nĂšse du syndrome neurodĂ©gĂ©nĂ©ratif SCAN1, qui est causĂ© par une dĂ©ficience en Tdp1
DNA-PK triggers histone ubiquitination and signaling in response to DNA double-strand breaks produced during the repair of transcription-blocking topoisomerase I lesions
Although defective repair of DNA double-strand breaks (DSBs) leads to neurodegenerative diseases, the processes underlying their production and signaling in non-replicating cells are largely unknown. Stabilized topoisomerase I cleavage complexes (Top1cc) by natural compounds or common DNA alterations are transcription-blocking lesions whose repair depends primarily on Top1 proteolysis and excision by tyrosyl-DNA phosphodiesterase-1 (TDP1). We previously reported that stabilized Top1cc produce transcription-dependent DSBs that activate ATM in neurons. Here, we use camptothecin (CPT)-treated serum-starved quiescent cells to induce transcription-blocking Top1cc and show that those DSBs are generated during Top1cc repair from Top1 peptide-linked DNA single-strand breaks generated after Top1 proteolysis and before excision by TDP1. Following DSB induction, ATM activates DNA-PK whose inhibition suppresses H2AX and H2A ubiquitination and the later assembly of activated ATM into nuclear foci. Inhibition of DNA-PK also reduces Top1 ubiquitination and proteolysis as well as resumption of RNA synthesis suggesting that DSB signaling further enhances Top1cc repair. Finally, we show that co-transcriptional DSBs kill quiescent cells. Together, these new findings reveal that DSB production and signaling by transcription-blocking Top1 lesions impact on non-replicating cell fate and provide insights on the molecular pathogenesis of neurodegenerative diseases such as SCAN1 and AT syndromes, which are caused by TDP1 and ATM deficiency, respectively
Dynamic Effects of Topoisomerase I Inhibition on R-Loops and Short Transcripts at Active Promoters.
Topoisomerase I-DNA-cleavage complexes (Top1cc) stabilized by camptothecin (CPT) have specific effects at transcriptional levels. We recently reported that Top1cc increase antisense transcript (aRNAs) levels at divergent CpG-island promoters and, transiently, DNA/RNA hybrids (R-loop) in nuclear and mitochondrial genomes of colon cancer HCT116 cells. However, the relationship between R-loops and aRNAs was not established. Here, we show that aRNAs can form R-loops in N-TERA-2 cells under physiological conditions, and that promoter-associated R-loops are somewhat increased and extended in length immediately upon cell exposure to CPT. In contrast, persistent Top1ccs reduce the majority of R-loops suggesting that CPT-accumulated aRNAs are not commonly involved in R-loops. The enhancement of aRNAs by Top1ccs is present both in human colon cancer HCT116 cells and WI38 fibroblasts suggesting a common response of cancer and normal cells. Although Top1ccs lead to DSB and DDR kinases activation, we do not detect a dependence of aRNA accumulation on ATM or DNA-PK activation. However, we showed that the cell response to persistent Top1ccs can involve an impairment of aRNA turnover rather than a higher synthesis rate. Finally, a genome-wide analysis shows that persistent Top1ccs also determine an accumulation of sense transcripts at 5'-end gene regions suggesting an increased occurrence of truncated transcripts. Taken together, the results indicate that Top1 may regulate transcription initiation by modulating RNA polymerase-generated negative supercoils, which can in turn favor R-loop formation at promoters, and that transcript accumulation at TSS is a response to persistent transcriptional stress by Top1 poisoning
DNA-PK triggers histone ubiquitination and signaling in response to DNA double-strand breaks produced during the repair of transcription-blocking topoisomerase I lesions
RNase H2, mutated in AicardiâGoutiĂšres syndrome, resolves co-transcriptional R-loops to prevent DNA breaks and inflammation
RNase H2 is a specialized enzyme that degrades RNA in RNA/DNA hybrids and deficiency of this enzyme causes a severe neuroinflammatory disease, Aicardi GoutiĂšres syndrome (AGS). However, the molecular mechanism underlying AGS is still unclear. Here, we show that RNase H2 is associated with a subset of genes, in a transcription-dependent manner where it interacts with RNA Polymerase II. RNase H2 depletion impairs transcription leading to accumulation of R-loops, structures that comprise RNA/DNA hybrids and a displaced DNA strand, mainly associated with short and intronless genes. Importantly, accumulated R-loops are processed by XPG and XPF endonucleases which leads to DNA damage and activation of the immune response, features associated with AGS. Consequently, we uncover a key role for RNase H2 in the transcription of human genes by maintaining R-loop homeostasis. Our results provide insight into the mechanistic contribution of R-loops to AGS pathogenesis
Dual Processing of R-Loops and Topoisomerase I Induces Transcription-Dependent DNA Double-Strand Breaks
International audienc
CPT modulates sense transcription in a particular subset of genes.
<p>Sense tags distribution along non-overlapping Refseq genes of HCT116 cells and HCT116-shRNATop1 cells, were analyzed in a region from 2000 bases upstream the TSS to 2000 bases downstream the TES using NGSplot software. Here, genes have been divided in two groups based on their FPKM. These genes have been selected for an accumulation of sense reads in the 5â region (CPT-reads minus Control-reads: 10 â„ 100) and a reduction of sense reads at the 3â region (CPT-reads minus Control-reads: †5). Furthermore, genes selected have a fold change above 2 and a minimum number of sense reads above 5 in the 5â region of CPT treated sample. Reads were normalized to the length of each region (1000 bp). Control reads are reported in gray dotted line and CPT reads in black line. Black arrows indicate an accumulation or not of reads at 5â level after CPT treatment (10 ÎŒM for 4 hours).</p