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

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE137685 | GEO | 2019/09/28

REPOSITORIES: GEO

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

2019-09-28 | GSE136943 | GEO

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

2019-09-28 | GSE136675 | 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

Reconstitution of SPO11-dependent double-strand break formation

Project description:Homologous meiotic recombination starts with DNA double-strand breaks (DSBs) generated by SPO11 protein. SPO11 is critical for meiosis in most species but the DSBs it makes are also dangerous because of their mutagenic and gametocidal potential, so cells must foster beneficial functions of SPO11 while minimizing its risks. SPO11 mechanism and regulation remain poorly understood. Here we report reconstitution of DNA cleavage in vitro with purified recombinant mouse SPO11 bound to its essential partner TOP6BL. Similar to their yeast orthologs, SPO11–TOP6BL complexes are monomeric (1:1) in solution and bind tightly to DNA. Unlike in yeast, however, dimeric (2:2) assemblies of mouse SPO11–TOP6BL cleaves DNA to form covalent 5 prime attachments requiring SPO11 active site residues, divalent metal ions, and SPO11 dimerization. Surprisingly, SPO11 can also manifest topoisomerase activity by relaxing supercoils and resealing DNA that it has nicked. Structure modeling with AlphaFold3 illuminates the protein-DNA interface and suggests that DNA is bent prior to cleavage. Deep sequencing of in vitro cleavage products reveals a rotationally symmetric base composition bias that partially explains DSB site preferences in vivo. Cleavage is inefficient on complex DNA substrates, partly because SPO11 is readily trapped in DSB-incompetent (presumably monomeric) binding states that exchange slowly. However, cleavage is improved by using substrates that favor DSB-competent dimer assembly, or by fusing SPO11 to an artificial dimerization module. Our results inform a model in which intrinsically feeble dimerization restrains SPO11 activity in vivo, making it exquisitely dependent on accessory proteins that focus and control DSB formation so that it happens only at the right time and the right places.

2024-08-20 | GSE275291 | 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

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

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 | GSE171042 | GEO

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

2021-06-09 | GSE169760 | GEO

Fun30-Myc ChIP-seq in Spo11 wild type and spo11-yf mutants

Project description:Resection, nucleolytic processing of DSB ends is necessary to generate 3' single-stranded DNA tails (ssDNA) for homologous recombination (HR). Meiotic recombination initiated by Spo11 induced double-strand breaks (DSBs) is essential for the accurate segregation of homologous chromosomes. After cleavage, Spo11 stays covalently linked to the DSB ends, which requires MRX/Sae2 incision on broken molecules to allow following Exo1-mediated resection. Exonuclease I activity is inhibited by nucleosome bound DNA in vitro. To ensure resection proceeds, resection machineries must overcome chromatin barrier. Here we show that DSB dependent enrichment of Fun30 proteins at hotspots and meiotic axes.

2023-05-01 | GSE221033 | GEO

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

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 | GSE129527 | GEO

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