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Topoisomerase I poisoning induces R-loop changes and DNA damage in human cancer cells [END-seq]

ABSTRACT: DNA topoisomerase I is an essential enzyme in higher eukaryotes that regulates DNA torsional tension during fundamental processes, such as replication and transcription. During its catalytic activity, a transient Topoisomerase I-DNA cleavage complex, called Top1cc, forms to allow strand rotation and duplex relaxation. However, the stabilization of Top1cc can lead to increased DNA:RNA hybrid duplexes, DNA double-strand cuts (DSBs) and genome instability as shown by formation of micronuclei, extranuclear chromatin enveloped by a nuclear membrane. To better comprehend the underlying mechanisms, we have established genomic maps of Top1cc-mediated R-loop changes and DSBs at short times upon Top1cc induction. Our findings show that Top1ccs dynamically changed the genomic distribution of R-loops, with regions of stable hybrid gains. DSBs were at specific gene loci, strongly associated with stable anterior R-loops at highly-transcribed genes and close to backtracked RNA polymerase II. Moreover, DSBs and stable anterior R-loops were highly enriched at early replication origins, thus revealing the location of replication conflicts with backtracked RNA polymerases.

ORGANISM(S): Homo sapiens

PROVIDER: GSE239327 | GEO | 2024/06/05

REPOSITORIES: GEO

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Topoisomerase I poisoning induces R-loop changes and DNA damage in human cancer cells [DRIP-seq]

Project description:DNA topoisomerase I is an essential enzyme in higher eukaryotes that regulates DNA torsional tension during fundamental processes, such as replication and transcription. During its catalytic activity, a transient Topoisomerase I-DNA cleavage complex, called Top1cc, forms to allow strand rotation and duplex relaxation. However, the stabilization of Top1cc can lead to increased DNA:RNA hybrid duplexes, DNA double-strand cuts (DSBs) and genome instability as shown by formation of micronuclei, extranuclear chromatin enveloped by a nuclear membrane. To better comprehend the underlying mechanisms, we have established genomic maps of Top1cc-mediated R-loop changes and DSBs at short times upon Top1cc induction. Our findings show that Top1ccs dynamically changed the genomic distribution of R-loops, with regions of stable hybrid gains. DSBs were at specific gene loci, strongly associated with stable anterior R-loops at highly-transcribed genes and close to backtracked RNA polymerase II. Moreover, DSBs and stable anterior R-loops were highly enriched at early replication origins, thus revealing the location of replication conflicts with backtracked RNA polymerases.

2024-06-05 | GSE239326 | GEO

R-loops and Topoisomerase 1 facilitate formation of transcriptional DSBs at gene bodies of hypertranscribed cancer genes

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2024-05-22 | GSE241309 | GEO

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2024-05-22 | GSE241305 | GEO

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2024-09-03 | PXD046037 | Pride

TEX264 drives selective autophagy of DNA lesions to promote DNA repair and cell survival

Project description:DNA repair and autophagy are distinct biological processes vital for cell survival. Although autophagy helps maintain genome stability, there is no evidence of its direct role in the repair of DNA lesions. We discovered that in human cells, lysosomes process Topoisomerase 1-cleavage complexes (TOP1cc) DNA lesion. Selective degradation of TOP1cc by autophagy directs DNA damage repair and cell survival at clinically relevant doses of Topoisomerase 1 inhibitors. TOP1cc are exported from the nucleus to lysosomes through transient alteration of the nuclear envelope, and independent of the proteasome. Mechanistically, the autophagy receptor TEX264 acts as a TOP1cc sensor at DNA replication forks, triggering TOP1cc processing by the p97 ATPase and mediating TOP1cc delivery to lysosomes dependent on MRE11 nuclease and ATR kinase. We found an evolutionary conserved role for selective autophagy in DNA damage repair that enables cell survival, protects genome stability, and is clinically relevant for colorectal cancer patients.

2024-09-01 | GSE242298 | GEO

Topoisomerase 1 prevents replication stress at R-loop-enriched transcription termination sites

Project description:R-loops have both positive and negative impacts on chromosome functions. To identify toxic R-loops in the human genome, we have mapped RNA:DNA hybrids, replication stress markers and DNA double strand breaks (DSBs) in cells depleted for Topoisomerase I (Top1), an enzyme that relaxes DNA supercoiling and prevents R-loop formation. RNA:DNA hybrids were found at both promoters (TSS) and terminators (TTS) of highly expressed genes. In contrast, the phosphorylation of RPA by ATR was only detected at TTS, which are preferentially replicated in a head-on orientation relative to the direction of transcription. In Top1-depleted cells, DSBs also cumulated at TTS, leading to persistent checkpoint activation, spreading of -H2AX on chromatin and global replication fork slowdown. These data indicate that fork pausing at the TTS of highly expressed genes containing R-loops prevents head-on conflicts between replication and transcription and maintains genome integrity in a Top1-dependent manner.

2020-06-20 | GSE108172 | GEO

TEX264 coordinates p97- and SPRTN-mediated resolution of topoisomerase 1-DNA adducts

Project description:Eukaryotic topoisomerase 1 (TOP1) regulates DNA topology to ensure efficient DNA replication and transcription. TOP1 is also a major driver of endogenous genome instability, particularly when its catalytic intermediate - a covalent TOP1-DNA adduct known as a TOP1 cleavage complex (TOP1cc) - is stabilised. TOP1ccs are highly cytotoxic and a failure to resolve them underlies the pathology of neurological disorders but is also exploited in cancer therapy where TOP1ccs are the target of widely used frontline anti-cancer drugs. A critical enzyme for TOP1cc resolution is the tyrosyl-DNA phosphodiesterase, TDP1, which hydrolyses the bond that links a tyrosine in the active site of TOP1 to a 3’ phosphate group on a single-stranded (ss)DNA break. However, TDP1 can only process small peptide fragments from ssDNA ends, raising the question of how the ~90 kDa TOP1 protein is processed upstream of TDP1. We find that TEX264 fulfils this role by forming a complex with the p97 ATPase and the SPRTN metalloprotease. We show that TEX264 recognises both unmodified and SUMO1-modifed TOP1 and initiates TOP1cc repair by recruiting p97 and SPRTN. TEX264 localises to the nuclear periphery, associates with DNA replication forks, and counteracts TOP1ccs during DNA replication. Altogether, our study elucidates the existence of a specialised repair complex required for upstream proteolysis of TOP1ccs and their subsequent resolution.

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Effect of TOP1 knockdown and/or RNase H OE on the transcriptional profile of MCF-7 cells [CEL_Seq]

Project description:DNA double-stranded breaks (DSBs) pose a significant threat to genomic integrity, and their generation during essential cellular processes like transcription remains poorly understood. In this study, we employed CEL-seq technique to investigate the effect of TOP1 KD and RNase H OE on transcriptional profile, with the different genetic manipulations. Our findings revealed the presence of DSBs at highly expressed genes enriched with TOP1 and R-loops, indicating their crucial involvement in transcription-associated genomic instability. Depletion of R-loops and TOP1 specifically reduced DSBs at highly expressed genes, uncovering their pivotal roles in transcriptional DSB formation. By elucidating the intricate interplay between TOP1cc trapping, R-loops, and DSBs, our study provides novel insights into the mechanisms underlying transcription-associated genomic instability. Moreover, we establish a link between transcriptional DSBs and early molecular changes driving cancer development. Notably, our study highlights the distinct etiology and molecular characteristics of driver mutations compared to passenger mutations, shedding light on the potential for targeted therapeutic strategies. Overall, these findings deepen our understanding of the regulatory mechanisms governing DSBs in hypertranscribed genes associated with carcinogenesis, opening avenues for future research and therapeutic interventions.

2024-05-22 | GSE241306 | GEO

Affinity Purification experiments for 24 human Bromodomain proteins involved in DNA repair and genome integrity (part 2 of 2)

Project description:Bromodomain proteins (BRD) are key chromatin regulators of genome function and stability, as well as therapeutic targets in cancer. In our associated publication (doi:10.1101/gad.331231.119) we systematically delineate the contribution of human BRD proteins for genome stability and DNA double-strand break (DSB) repair using cell-based assays and proteomic interaction network analysis. These AP-MS experiments were performed in order to construct a protein interaction network for the 24 BRDs we identified as promoters of DNA repair and/or genome integrity: BAZ1B, BRD1, BRD2, BRD3, BRD4, BRD8, BRD9, BRPF3, BRWD3, CECR2, EP300, GCN5/KAT2A, PCAF/KAT2B, PHIP, SMARCA2, SP100, SP110, SP140, TAF1, TRIM24, TRIM28, TRIM33, TRIM66, ZMYND8. In combination with cell based assays, we identified a BRD-reader function of PCAF that bound TIP60-mediated histone acetylations at DSBs to recruit a DUB complex to deubiquitylate histone H2BK120, to allow direct acetylation by PCAF and repair of DSBs by homologous recombination. We also discovered the bromo-and-extra-terminal (BET) BRD proteins, BRD2 and BRD4, as negative regulators of transcription-associated RNA-DNA hybrid (R-Loop) as inhibition of BRD2 or BRD4 increased R-loop formation, which generated DSBs. These breaks were reliant on Topoisomerase II and BRD2 directly bound and activated Topoisomerase I, a known restrainer of R-loops. Thus, comprehensive interactome and functional profiling of BRD proteins revealed new homologous recombination and genome stability pathways, providing a strategy to understand genome maintenance by BRD proteins and the effects of their pharmacological inhibition. This accession provides AP-MS experiment files for the following subset of 6 BRDs: BRD9, EP300, GCN5/KAT2A, PCAF/KAT2B, SP140, TRIM28, as well as their associated controls (Input, Mock, and SBP-tagged NLS).

2019-12-12 | PXD016673 | Pride

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