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Nucleotide resolution mapping of Top2-linked DNA breaks in the human genome

ABSTRACT: DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

ORGANISM(S): Homo sapiens

PROVIDER: GSE136943 | GEO | 2019/09/28

REPOSITORIES: GEO

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Nucleotide resolution mapping of Top2-linked DNA breaks in the yeast genome

Project description:DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes—and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy reveals the influence primary DNA sequence has upon Top2 cleavage—distinguishing sites likely to form canonical DNA double-strand breaks (DSBs) from those predisposed to form strand-biased DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo. This data set contains maps of Top2 CCs in the S. cerevisiae genome, generated by CC-seq of BY4741 cells -/+ etoposide (VP16).

2019-09-28 | GSE136675 | GEO

Nucleotide resolution mapping of Spo11-linked DNA breaks in the yeast genome

Project description:DNA topoisomerases are required to resolve DNA topological stress. Despite this essential role, abortive topoisomerase activity generates aberrant protein-linked DNA breaks, jeopardising genome stability. Here, to understand the genomic distribution and mechanisms underpinning topoisomerase-induced DNA breaks, we map Top2 DNA cleavage with strand-specific nucleotide resolution across the S. cerevisiae and human genomes - and use the meiotic Spo11 protein to validate the broad applicability of this method to explore the role of diverse topoisomerase family members. Our data characterises Mre11-dependent repair in yeast, and defines two strikingly different fractions of Top2 activity in humans: tightly localised CTCF-proximal, and broadly distributed transcription-proximal, the latter correlated with gene length and expression. Moreover, single nucleotide accuracy enables us to reveal the influence primary DNA sequence has upon Top2 cleavage - distinguishing canonical DNA double-strand breaks (DSBs) from a major population of DNA single-strand breaks (SSBs) induced by etoposide (VP16) in vivo.

2019-09-28 | GSE137685 | GEO

RAD54L2 mediates a novel mechanism to counter TOP2-DNA adducts

Project description:The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anti-cancer agents such as etoposide operate by stabilising TOP2ccs, ultimately generating genotoxic TOP2-DNA protein crosslinks that require processing and repair. Here, we identify RAD54L2 as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with ZNF451 and independent of TDP2. Our work suggests a model wherein RAD54L2 recognises sumoylated-TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer-therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.

2024-01-26 | PXD046611 | Pride

ATM suppresses c-Myc overexpression in the mammary epithelium in response to estrogen

Project description:ATM gene mutation carriers are predisposed to estrogen receptor (ER)-positive breast cancer (BC). ATM prevents BC oncogenesis by activating p53 in every cell; however, much remains unknown about tissue-specific oncogenesis following ATM loss. Here, we report that ATM controls the early transcriptional response to estrogens. This response depends on topoisomerase II (TOP2), which generates TOP2-DNA double-strand break (DSB) complexes and rejoins the breaks. When TOP2-mediated ligation fails, ATM facilitates DSB repair. Following estrogen exposure, TOP2-dependent DSBs arise at the c-MYC enhancer in human BC cells, and their defective repair changes the activation profile of enhancers and induces the overexpression of many genes including the c-MYC oncogene. CRISPR/Cas9 cleavage at the enhancer also causes c-MYC overexpression, indicating that this DSB causes c-MYC overexpression. Estrogen treatment induced c-Myc protein overexpression in mammary epithelial cells of ATM-deficient mice. In conclusion, ATM suppresses the c-Myc-driven proliferative effects of estrogens, possibly explaining such tissue-specific oncogenesis.

2023-06-01 | GSE218320 | GEO

Genome wide binding of Top2 (in WT and uls1 delta cells) and Uls1 (in WT cells) both in the presence and absence of the Top2 poison acriflavine (ACF)

Project description:Topoisomerase II (Top2) is an essential enzyme that decatenates DNA via a transient Top2-DNA covalent intermediate. This intermediate can be stabilised by a class of drugs termed Top2 poisons, resulting in massive DNA damage. Thus, Top2 activity is a double-edged sword that needs to be carefully controlled to maintain genome stability. We show that Uls1, an ATP-dependent chromatin remodelling (Snf2) enzyme, can alter Top2 chromatin binding and prevent Top2 poisoning in yeast. Deletion mutants of ULS1 are hypersensitive to the Top2 poison acriflavine (ACF), activating the DNA damage checkpoint. We map Uls1’s Top2 interaction domain and show that this, together with its ATPase activity, is essential for Uls1 function. By performing ChIP-seq, we show that ACF leads to a general increase in Top2 binding across the genome. We map Uls1 binding sites and identify tRNA genes as key regions where Uls1 associates after ACF treatment. Importantly, the presence of Uls1 at these sites prevents ACF-dependent Top2 accumulation. Our data reveal the effect of Top2 poisons on the global Top2 binding landscape and highlights the role of Uls1 in antagonising Top2 function. Remodelling Top2 binding is thus an important new means by which Snf2 enzymes promote genome stability.

2019-04-17 | GSE123707 | GEO

Spo11 generates gaps through concerted cuts at sites of topological stress [Top2]

Project description:Meiotic recombination is essential for proper meiotic chromosome segregation and fertility, and is initiated by programmed DNA double-strand breaks (DSBs) introduced by Spo11, a eukaryotic homolog of archaeal topoisomerase VIA. Here we report the discovery of hitherto uncharacterized Spo11-induced lesions, small gaps from 34 bp to several hundred bp, which are generated by coordinated pairs of DSBs (double DSBs or dDSBs). Isolation and genome-wide mapping of the resulting fragments with single base pair precision reveals enrichment at DSB hotspots but also a widely dispersed distribution covering the entire genome. We show that Spo11 prefers to cut at a sequence motif which promotes DNA bending, indicating that bendability of DNA contributes to cleavage site choice. Moreover, fragment lengths display a ~ (10.4n+3) bp periodicity, implying that Spo11 favours cleavage on the same face of underwound DNA. Consistently, dDSB signals overlap and correlate with topoisomerase II binding sites, which points to a role for topological stress and DNA crossings in break formation, and suggests a unified model for DSB and dDSB formation, in which Spo11 traps two DNA strands. Furthermore, gaps resulting from dDSBs, an estimated 20% of all initiation events, can account for full gene conversion events that are independent of both Msh2-dependent heteroduplex repair and MutLγ. Since non-homologous gap repair results in deletions, and ectopically re-integrated dDSB fragments result in insertions, dDSB formation represents a potential source of evolutionary diversity and pathogenic germ-line aberrations.

2021-06-09 | GSE169760 | GEO

Hotspots of single-strand DNA "breakome" are enriched at transcriptional start sites of genes

Project description:Single strand breaks (SSBs) represent one of the most common types of DNA damage yet not much is known about the genome landscapes of this type of DNA lesions in mammalian cells. Here, we found that SSBs are more likely to occur in certain positions of the human genome — SSB hotspots — in different cells of the same cell type and in different cell types. We hypothesize that the hotspots are likely to represent biologically relevant breaks. And, we found that the hotspots had a prominent tendency to be enriched in the immediate vicinity of transcriptional start sites (TSSs). We show that these hotspots are not likely to represent technical artifacts, or be caused by common mechanisms previously found to cause DNA cleavage at promoters, such as apoptotic DNA fragmentation or topoisomerase type II (TOP2) activity. Therefore, such TSS-associated hotspots could potentially be generated using a novel mechanism, that could involve preferential cleavage at cytosines, and their existence is consistent with recent studies suggesting a complex relationship between DNA damage and regulation of gene expression.

2022-08-18 | GSE190735 | GEO

Effect of top2 and hmo1 on chromatin architecture and transcription in yeast

Project description:DNA topoisomerase-2 and high mobility group protein Hmo1 are known to regulate chromatin architecture by regulating gene boundaries. Here we report how these proteins affect global RNA level after inactivation of Top2 and Hmo1. Our data indicate that inactivating Hmo1 has a drastic effect on transcription levels of 20% yeast genes, however, this phenomenon can slightly be rescued by inactivating Top2 functions. Also, we study the Top2 and Top1 role in nucleosome architecture with and without expressing E.coli TopA. In top2-1;top1∆ condition with TopA expressed, it affects the nucleosome occupancy at the global level compared with top2-1;top1∆-Control plasmid. The ChIA-PET (Chromatin interaction analysis by paired-end tag sequencing) method is used to address whether a specific protein is engaged in the chromosomal interactions. Epitope tagged Top2 protein is used as probe in ChIA-PET experiments to map Top2 mediated chromatin-chromatin interactions.

2018-07-02 | GSE114444 | GEO

Replication termination is mediated by Top2 and occurs at genomic loci containing pausing elements

Project description:[original title] Chromosome replication initiates at multiple replicons and terminates when forks converge. In Escherichia coli, the Tus-TER complex mediates polar fork converging at the terminator region and aberrant termination events challenge chromosome integrity and segregation. Since in eukaryotes termination is less characterized, we used budding yeast to identify the factors assisting fork fusion at replicating chromosomes. Using genomic and mechanistic studies we have identified and characterized 71 chromosomal termination regions (TERs). TERs contain fork pausing elements that influence fork progression and merging. The Rrm3 DNA helicase assists fork progression across TERs counteracting the accumulation of X-shaped structures. The Top2 DNA topoisomerase associates at TERs in S-phase and G2/M facilitates fork fusion and prevents DNA breaks and genome rearrangements at TERs. We propose that in eukaryotes replication fork barriers, Rrm3 and Top2 coordinate replication fork progression and fusion at termination regions thus counteracting abnormal genomic transitions. Signal tracks in BED format suitable for visualization on the UCSC genome browser can be found at http://bio.ifom-ieo-campus.it/supplementary/Fachinetti_et_al_MOLCELL_2010

2010-08-28 | GSE19061 | GEO

Topoisomerase II-induced Chromosome Breakage and Translocation Is Determined by Chromosome Architecture and Transcriptional Activity [HTGTS]

Project description:Topoisomerase II (TOP2) relieves torsional stress during transcription, DNA replication and chromosome segregation, by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks. While TOP2ccs are normally reversible they can be ‘trapped’ by chemotherapeutic drugs such as etoposide, and subsequently converted into irreversible TOP2-linked DSBs that threaten genome stability. Here, using genomics approaches, we have quantified the etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 trapping is independent of transcription it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated two-fold at transcribed loci, relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two critical features of pre-existing chromatin structure- namely, cohesin binding and transcriptional activity- can be used to accurately predict the kinetics of TOP2-induced DSBs. Thus, our study permits a mechanistic understanding of TOP2 induced genome instability.

2019-04-26 | GSE129528 | GEO

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