Novel approach reveals genomic landscapes of single-strand DNA breaks with nucleotide resolution in human cells

Cao, Huifen; Salazar-García, Lorena; Gao, Fan; Wahlestedt, Thor; Wu, Chun Lin; Han, Xueer; Cai, Ye; Xu, Dongyang; Wang, Fang; Tang, Lu; Ricciardi, Natalie; Cai, Ding Ding; Wang, Hong; Chin, Mario P. S.; Timmons, James A.; Wahlestedt, Claes; Kapranov, Philipp

doi:10.1038/s41467-019-13602-7

Cited by 37 publications

(50 citation statements)

References 54 publications

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“…The other method, SSiNGLe, is quite different from Nick-seq and is limited solely to mapping single-strand breaks with 3′-hydroxyl groups ( 36 ). Unfortunately, the method cannot be applied to any other form of DNA modification or damage and cannot be used for single-strand breaks with ‘dirty 3′-ends’ as occur in DNA repair intermediates (e.g.…”

Section: Discussionmentioning

confidence: 99%

“…In fact, the DNA fragmentation with micrococcal nuclease relies on the presence of nucleosomes to limit the fragmentation to the ∼150 nt periodicity of nucleosome-bound DNA ( 40 ). Furthermore, the accuracy of SSiNGLe for single-nucleotide resolution is relatively low, with only 87–95% of the breaks mapping to ± 1 nt of the true cutting site of the single-strand endonuclease Nt.BbvCI used to validate the method ( 36 ), with no mention of the number of missed consensus sites. The combination of NT and TdT labelling in Nick-seq significantly increases the accuracy of the method, with 98% of the exact cutting sites accurately called.…”

Section: Discussionmentioning

confidence: 99%

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Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage

Cao

Zhou

et al. 2020

Nucleic Acids Research

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Abstract DNA damage and epigenetic marks are well established to have profound influences on genome stability and cell phenotype, yet there are few technologies to obtain high-resolution genomic maps of the many types of chemical modifications of DNA. Here we present Nick-seq for quantitative, sensitive, and accurate mapping of DNA modifications at single-nucleotide resolution across genomes. Pre-existing breaks are first blocked and DNA modifications are then converted enzymatically or chemically to strand-breaks for both 3′-extension by nick-translation to produce nuclease-resistant oligonucleotides and 3′-terminal transferase tailing. Following library preparation and next generation sequencing, the complementary datasets are mined with a custom workflow to increase sensitivity, specificity and accuracy of the map. The utility of Nick-seq is demonstrated with genomic maps of site-specific endonuclease strand-breaks in purified DNA from Eschericia coli, phosphorothioate epigenetics in Salmonella enterica Cerro 87, and oxidation-induced abasic sites in DNA from E. coli treated with a sublethal dose of hydrogen peroxide. Nick-seq applicability is demonstrated with strategies for >25 types of DNA modification and damage.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage

Cao

Zhou

et al. 2020

Nucleic Acids Research

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“…To date, four NGS-based methods have been developed for the genome-wide mapping of SSBs: SSB-Seq [71,72], Nick-Seq [73], SSiNGLe [74] and GLOE-Seq [75] (Table 1). Although the molecular steps involved in generating sequencing libraries vary, they all rely on the common principle of detecting SSBs by capturing free 3 0 -OH termini.…”

Section: Ngs-based Methods For Genome-wide Mapping Of Ssbsmentioning

confidence: 99%

Exploring the SSBreakome: genome‐wide mapping of DNA single‐strand breaks by next‐generation sequencing

Zilio

Ulrich

2020

The FEBS Journal

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Mapping the genome-wide distribution of DNA lesions is key to understanding damage signalling and DNA repair in the context of genome and chromatin structure. Analytical tools based on high-throughput next-generation sequencing have revolutionized our progress with such investigations, and numerous methods are now available for various base lesions and modifications as well as for DNA double-strand breaks. Considering that single-strand breaks are by far the most common type of lesion and arise not only from exposure to exogenous DNA-damaging agents, but also as obligatory intermediates of DNA replication, recombination and repair, it is surprising that our insight into their genome-wide patterns, that is the 'SSBreakome', has remained rather obscure until recently, due to a lack of suitable mapping technology. Here we briefly review classical methods for analysing single-strand breaks and discuss and compare in detail a series of recently developed high-resolution approaches for the genome-wide mapping of these lesions, their advantages and limitations and how they have already provided valuable insight into the impact of this type of damage on the genome.

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“…Direct ligation of a sequencing adaptor to the 3’ end of individual DNA strands would be a very attractive means of quantifying DNA damage irrespective of DNA resection, and direct labelling of DNA 3’ ends may reveal replication fork direction, particularly in mutants unable to ligate Okazaki fragments. Some methods aimed at mapping single-strand breaks and base changes theoretically have this capability although they have not been applied to DSBs [34, 35], and very recently the Ulrich lab described such a method, GLOE-seq, that is capable of replication profiling in DNA ligase-deficient yeast and human cells, and also maps DSBs although activity on resected substrates was not tested [36]. Here we describe an alternative method, T ransferase A ctivated E nd L igation sequencing (TrAEL-seq), which accurately maps DNA 3’ ends at DSBs that have undergone DNA resection.…”

Section: Introductionmentioning

confidence: 99%

Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

Kara

Krueger

Rugg‐Gunn

et al. 2020

Preprint

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Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods sensitive to both changes in replication fork structure and the formation of recombinogenic DNA ends. Here we describe Tr ansferase- A ctivated E nd L igation seq uencing (TrAEL-seq), a method that captures single stranded DNA 3’ ends genome-wide and with base pair resolution. TrAEL-seq labels DNA breaks, and profiles both stalled and processive replication forks in yeast and mammalian cells. Replication forks stalling at defined barriers and expressed genes are detected by TrAEL-seq with exceptional signal-to-noise, most likely through labelling of DNA 3’ ends exposed during fork reversal. TrAEL-seq also labels unperturbed processive replication forks to yield maps of replication fork direction similar to those obtained by Okazaki fragment sequencing, however TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3’ ends also allows accurate detection of double strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double strand break hotspots in a dmc1 Δ mutant. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

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Novel approach reveals genomic landscapes of single-strand DNA breaks with nucleotide resolution in human cells

Cited by 37 publications

References 54 publications

Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage

Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage

Exploring the SSBreakome: genome‐wide mapping of DNA single‐strand breaks by next‐generation sequencing

Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

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