Quantification and genome-wide mapping of DNA double-strand breaks

Grégoire, Marie-Chantal; Massonneau, Julien; Leduc, Frédéric; Arguin, Mélina; Brazeau, Marc-André; Boissonneault, Guylain

doi:10.1016/j.dnarep.2016.10.005

Cited by 19 publications

(12 citation statements)

References 22 publications

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“…Recently, the TUNEL assay was used to demonstrate the cytotoxic effect caused by the release of the aptamer‐incorporated drug doxorubicin . The TUNEL method has also been employed for the genome‐wide mapping, at nucleotide‐level resolution, of double‐strand breaks in yeast and HeLa cells . Given the importance and popularity of the TUNEL assay, kits are now commercially available from different suppliers for the in situ detection of apoptosis (e.g., TACS kit).…”

Section: Incorporation Of Modified Nucleotidesmentioning

confidence: 99%

“…[77] The TUNEL methodh as also been employed for the genome-wide mapping, at nucleotide-level resolution, of double-strand breaks in yeast and HeLa cells. [68,78] Given the importance and popularity of the TUNEL assay,k its are now commercially available from different suppliers for the in situ detection of apoptosis (e.g.,T ACS kit).…”

Section: Immobilization Of Dna and Tunel Assaymentioning

confidence: 99%

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Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

Sarac

Hollenstein

2019

ChemBioChem

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The terminal deoxynucleotidyl transferase (TdT) belongs to the X family of DNA polymerases. This unusual polymerase catalyzes the template‐independent addition of random nucleotides on 3′‐overhangs during V(D)J recombination. The biological function and intrinsic biochemical properties of the TdT have spurred the development of numerous oligonucleotide‐based tools and methods, especially if combined with modified nucleoside triphosphates. Herein, we summarize the different applications stemming from the incorporation of modified nucleotides by the TdT. The structural, mechanistic, and biochemical properties of this polymerase are also discussed.

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Section: Incorporation Of Modified Nucleotidesmentioning

confidence: 99%

Section: Immobilization Of Dna and Tunel Assaymentioning

confidence: 99%

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

Sarac

Hollenstein

2019

ChemBioChem

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“…The relative increase in mobility between samples is therefore expected to result from alterations in the repair of bleomycin‐induced DSBs. DNA 3'OH ends are expected to result from bleomycin action, so we determined the relative extent of 3'OH at DSBs using a modification of the TUNEL assay termed ‘qTUNEL’ allowing specific radioactive detection of DSBs following an initial step of nicks and gaps repair . As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A modification of the terminal deoxynucleotidyl transferase (TdT) nick‐end labeling method has been developed by our group allowing for the relative quantification of DSBs (qTUNEL) . Briefly, an initial step of DNA nick and gap repair is performed using a combination of T4 DNA ligase and T4 DNA polymerase prior to radioactive labeling using 0.13 m m of dATP, [α‐ 32 P], and 0.013 m m ddATP.…”

Section: Methodsmentioning

confidence: 99%

Suboptimal extracellular pH values alter DNA damage response to induced double‐strand breaks

et al. 2018

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Conditions leading to unrepaired DNA double‐stranded breaks are potent inducers of genetic instability. Systemic conditions may lead to fluctuation of hydrogen ions in the cellular microenvironment, and we show that small variations in extracellular pH , termed suboptimal pH e, can decrease the efficiency of DNA repair in the absence of intracellular pH variation. Recovery from bleomycin‐induced DNA double‐stranded breaks in fibroblasts proceeded less efficiently at suboptimal pH e values ranging from 7.2 to 6.9, as shown by the persistence of repair foci, reduction of H4K16 acetylation, and chromosomal instability, while senescence or apoptosis remained undetected. By allowing escape from these protective mechanisms, suboptimal pH e may therefore enhance the genotoxicity of double‐stranded breaks, leading to genetic instability.

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“…Quantitative Polymerase Chain Reaction (qPCR) based methods can estimate absolute break frequency but only at selected loci 11 . An approach was developed recently to quantify breaks globally based on amount of radiolabeled DNA and locally based on DNA break immunocapture 12 , but its accuracy in detecting physiological DSBs was not tested. BLISS 7 quantifies DSBs by utilizing unique molecular identifiers (UMIs) to identify unique DSB ends and counting cells in the sample.…”

Section: Introductionmentioning

confidence: 99%

qDSB-Seq: quantitative DNA double-strand break sequencing

Zhu

Biernacka

Pardo³

et al. 2017

Preprint

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Emerging genome-wide methods for mapping DNA double-strand breaks (DSBs) by sequencing (e.g. BLESS) are limited to measuring relative frequencies of breaks between loci. Knowing the absolute DSB frequency per cell, however, is key to understanding their physiological relevance. Here, we propose quantitative DSB sequencing (qDSBSeq), a method to infer the absolute DSB frequency genome-wide. qDSB-Seq relies on inducing spike-in DSBs by a site-specific endonuclease and estimating the efficiency of the endonuclease cleavage by sequencing or PCR. This spike-in frequency is used to quantify DSB sequencing data. We present validation of the qDSB-Seq method and results of its application. We quantify DSBs resulting from replication stress and the collapse of replication forks on natural fork barriers in the ribosomal DNA. The qDSBSeq approach can be used with any DSB sequencing method and allows accurate comparisons of absolute DSB frequencies across samples and precise quantification of the impact of various DSB-causing agents.peer-reviewed)

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Quantification and genome-wide mapping of DNA double-strand breaks

Cited by 19 publications

References 22 publications

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

Terminal Deoxynucleotidyl Transferase in the Synthesis and Modification of Nucleic Acids

Suboptimal extracellular pH values alter DNA damage response to induced double‐strand breaks

qDSB-Seq: quantitative DNA double-strand break sequencing

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