A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

Gittens, William; Johnson, Dominic; Allison, Rachal M.; Cooper, Tim J.; Thomas, Holly R.; Neale, Matthew J.

doi:10.1101/530667

Cited by 16 publications

(30 citation statements)

References 78 publications

(25 reference statements)

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“…Furthermore, the pattern of Ts1 distribution around TSS (shown in Supplementary Fig. S3 ) was very similar to that of DSB detected by END-seq in human cells 37 .…”

Section: Discussionsupporting

confidence: 67%

Topoisomerase IIβ targets DNA crossovers formed between distant homologous sites to induce chromatin opening

Miyaji

Furuta

Hosoya

et al. 2020

Sci Rep

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Type II DNA topoisomerases (topo II) flip the spatial positions of two DNA duplexes, called G- and T- segments, by a cleavage-passage-resealing mechanism. In living cells, these DNA segments can be derived from distant sites on the same chromosome. Due to lack of proper methodology, however, no direct evidence has been described so far. The beta isoform of topo II (topo IIβ) is essential for transcriptional regulation of genes expressed in the final stage of neuronal differentiation. Here we devise a genome-wide mapping technique (eTIP-seq) for topo IIβ target sites that can measure the genomic distance between G- and T-segments. It revealed that the enzyme operates in two distinctive modes, termed proximal strand passage (PSP) and distal strand passage (DSP). PSP sites are concentrated around transcription start sites, whereas DSP sites are heavily clustered in small number of hotspots. While PSP represent the conventional topo II targets that remove local torsional stresses, DSP sites have not been described previously. Most remarkably, DSP is driven by the pairing between homologous sequences or repeats located in a large distance. A model-building approach suggested that topo IIβ acts on crossovers to unknot the intertwined DSP sites, leading to chromatin decondensation.

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“…Furthermore, the pattern of Ts1 distribution around TSS (shown in Supplementary Fig. S3 ) was very similar to that of DSB detected by END-seq in human cells 37 .…”

Section: Discussionsupporting

confidence: 67%

Topoisomerase IIβ targets DNA crossovers formed between distant homologous sites to induce chromatin opening

Miyaji

Furuta

Hosoya

et al. 2020

Sci Rep

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“…Next, we examined the effect of TOP2B knockout in the generation of DNA breaks in human cells using the recent CC-seq data in RPE-1 cells by Gittens et al. ( 15 ), that maps TOP2 cleavage complex-associated DSBs at single-nucleotide resolution. We first identified DSB peaks from wild-type cells treated with etoposide, to indicate genomic regions targeted by TOP2 activity ( n = 65 989).…”

Section: Resultsmentioning

confidence: 99%

“…The CC-seq data from Gittens et al. ( 15 ) was downloaded as fastq files from (GSE136943) and then aligned to the human genome (build GRCh38/hg38) following the same processing as break data (as detailed above in ‘Processing of DSB reads’). The combined data from four replicates of VP16 (etoposide)-treated WT RPE-1 cells was merged into one bam file using samtools merge, and peaks were called (as detailed above in ‘Peak calling’) ( n = 65 989).…”

Section: Methodsmentioning

confidence: 99%

Topoisomerase II contributes to DNA secondary structure-mediated double-stranded breaks

Szlachta

Manukyan

Raimer

et al. 2020

Nucleic Acids Research

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Abstract DNA double-stranded breaks (DSBs) trigger human genome instability, therefore identifying what factors contribute to DSB induction is critical for our understanding of human disease etiology. Using an unbiased, genome-wide approach, we found that genomic regions with the ability to form highly stable DNA secondary structures are enriched for endogenous DSBs in human cells. Human genomic regions predicted to form non-B-form DNA induced gross chromosomal rearrangements in yeast and displayed high indel frequency in human genomes. The extent of instability in both analyses is in concordance with the structure forming ability of these regions. We also observed an enrichment of DNA secondary structure-prone sites overlapping transcription start sites (TSSs) and CCCTC-binding factor (CTCF) binding sites, and uncovered an increase in DSBs at highly stable DNA secondary structure regions, in response to etoposide, an inhibitor of topoisomerase II (TOP2) re-ligation activity. Importantly, we found that TOP2 deficiency in both yeast and human leads to a significant reduction in DSBs at structure-prone loci, and that sites of TOP2 cleavage have a greater ability to form highly stable DNA secondary structures. This study reveals a direct role for TOP2 in generating secondary structure-mediated DNA fragility, advancing our understanding of mechanisms underlying human genome instability.

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“…Recently, Gittens et al developed a method to specifically map genome-wide TOP2 cleavage complexes at single-nucleotide resolution [37]. The mechanism of TOP2 cleavage results in a transient 4-nt 5′ overhang with the TOP2 cleavage complex attached on the 5′ ends [38].…”

Section: Discussionmentioning

confidence: 99%

“…Raw fastq data (GSE136943) [37] was downloaded and aligned to the human genome (GRCh38/hg38) following the same alignment processing as our break mapping data ("Sequencing Data Analysis" above). Aligned data was then processed through our CNCC analysis ("Coverage-Normalized Cross Correlation" above) and visualized.…”

Section: Top2 Cleavage Complex Single-nucleotide Sequencing Read Promentioning

confidence: 99%

CNCC: an analysis tool to determine genome-wide DNA break end structure at single-nucleotide resolution

et al. 2020

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Background: DNA double-stranded breaks (DSBs) are potentially deleterious events in a cell. The end structures (blunt, 3′-and 5′-overhangs) at DSB sites contribute to the fate of their repair and provide critical information concerning the consequences of the damage. Therefore, there has been a recent eruption of DNA break mapping and sequencing methods that aim to map at single-nucleotide resolution where breaks are generated genomewide. These methods provide high resolution data for the location of DSBs, which can encode the type of endstructure present at these breaks. However, genome-wide analysis of the resulting end structures has not been investigated following these sequencing methods. Results: To address this analysis gap, we develop the use of a coverage-normalized cross correlation analysis (CNCC) to process the high-precision genome-wide break mapping data, and determine genome-wide break end structure distributions at single-nucleotide resolution. We take advantage of the single-nucleotide position and the knowledge of strandness from every mapped break to analyze the relative shifts between positive and negative strand encoded break nucleotides. By applying CNCC we can identify the most abundant end structures captured by a break mapping technique, and further can make comparisons between different samples and treatments. We validate our analysis with restriction enzyme digestions of genomic DNA and establish the sensitivity of the analysis using end structures that only exist as a minor fraction of total breaks. Finally, we demonstrate the versatility of our analysis by applying CNCC to the breaks resulting after treatment with etoposide and study the variety of resulting end structures. Conclusion: For the first time, on a genome-wide scale, our analysis revealed the increase in the 5′ to 3′ end resection following etoposide treatment, and the global progression of the resection. Furthermore, our method distinguished the change in the pattern of DSB end structure with increasing doses of the drug. The ability of this method to determine DNA break end structures without a priori knowledge of break sequences or genomic position should have broad applications in understanding genome instability.

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A nucleotide resolution map of Top2-linked DNA breaks in the yeast and human genome

Cited by 16 publications

References 78 publications

Topoisomerase IIβ targets DNA crossovers formed between distant homologous sites to induce chromatin opening

Topoisomerase IIβ targets DNA crossovers formed between distant homologous sites to induce chromatin opening

Topoisomerase II contributes to DNA secondary structure-mediated double-stranded breaks

CNCC: an analysis tool to determine genome-wide DNA break end structure at single-nucleotide resolution

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