Rapid Quantification of Oxidation and UV Induced DNA Damage by Repair Assisted Damage Detection-(Rapid RADD)

Gilat, Noa; Torchinsky, Dmitry; Margalit, Sapir; Michaeli, Yael; Avraham, Sigal; Sharim, Hila; Elkoshi, Nadav; Levy, Carmit; Zirkin, Shahar; Ebenstein, Yuval

doi:10.1021/acs.analchem.0c01393

Cited by 12 publications

(16 citation statements)

References 31 publications

(47 reference statements)

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“…We first evaluated the global DNA damage levels in the two samples using an optical method known as Rapid-RADD (Gilat et al 2020). The optical experiment showed a ~30% decrease in the global damage level in the repaired samples compared with the damaged samples, verifying that DNA is indeed repaired during the 1h experimental repair period ( Supplemental Fig.…”

Section: Resultsmentioning

confidence: 99%

“…DNA samples extracted for the RADD-seq protocol, were used for Rapid-RADD procedure as well as described by Gilat et al (Gilat et al 2020). In short, KBrO 3 -treated DNA samples were labeled for oxidative DNA damage as described above, where instead of adding biotinylated-dUTP, a fluorescent ATTO-550-UTP (Jena biosciences GMBH) in the same quantity was added in the nucleotide mixture.…”

Section: Methodsmentioning

confidence: 99%

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Genome wide mapping of DNA lesions by Repair Assisted Damage Detection sequencing – RADD-Seq

Gilat

Fridman

Sharim

et al. 2021

Preprint

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Mapping DNA damage and its repair has immense potential in understanding environmental exposures, their genotoxicity, and their impact on human health. Monitoring changes in genomic stability also aids in the diagnosis of numerous DNA-related diseases, like cancer, and assists in monitoring their progression and prognosis. However, genome-wide maps of DNA damage distribution are challenging to produce. Here we describe the localization of DNA damage and repair loci by Repair Assisted Damage Detection sequencing - RADD-Seq. Based on the enrichment of damage lesions coupled with a pull-down assay and followed by next generation sequencing, this method is easy to perform and can produce compelling results with minimal coverage. RADD-seq enables the localization of both DNA damage and repair sites for a wide range of single-strand damage types. Using this technique, we created a genome-wide map of oxidative DNA damage loci before and after repair. Oxidative lesions were heterogeneously distributed along the human genome, with less damage occurring in tight chromatin regions. Furthermore, we showed repair is prioritized for highly expressed, essential genes and in open chromatin regions. RADD-seq sheds light on cellular repair mechanisms and capable of identifying genomic hotspots prone to mutation.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Genome wide mapping of DNA lesions by Repair Assisted Damage Detection sequencing – RADD-Seq

Gilat

Fridman

Sharim

et al. 2021

Preprint

Self Cite

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show abstract

“…DNA samples extracted for the RADD-seq protocol, were used for the Rapid-RADD procedure as well, as described by Gilat et al. ( 16 ). In short, KBrO 3 -treated DNA samples were labeled for oxidation DNA damage as described above, but instead of adding biotinylated dUTP, a fluorescent ATTO-550-UTP (Jena Bioscience, Jena, Germany) in the same quantity was added in the nucleotide mixture.…”

Section: Methodsmentioning

confidence: 99%

“…In this assay, damaged lesions are repaired within the cells themselves, and by measuring the fluorescent intensity of the cell, an accurate assessment of the overall damage in the cell is obtained. By using the same biochemistry, we recently introduced a method for the bulk quantification of multiple samples over a single multiwell glass slide ( 16 ). In this method, termed rapid quantification of DNA damage by repair-assisted damage detection (Rapid-RADD), an absolute quantification of the DNA damage in a sample is easily obtained.…”

Section: Introductionmentioning

confidence: 99%

From single-molecule to genome-wide mapping of DNA lesions: repair-assisted damage detection sequencing

Gilat

Fridman

Sharim

et al. 2021

Biophysical Reports

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Mapping DNA damage and its repair has immense potential in understanding environmental exposures, their genotoxicity, and their impact on human health. Monitoring changes in genomic stability also aids in the diagnosis of numerous DNA-related diseases, such as cancer, and assists in monitoring their progression and prognosis. Developments in recent years have enabled unprecedented sensitivity in quantifying the global DNA damage dose in cells via fluorescence-based analysis down to the single-molecule level. However, genome-wide maps of DNA damage distribution are challenging to produce. Here, we describe the localization of DNA damage and repair loci by repair-assisted damage detection sequencing (RADD-seq). Based on the enrichment of damage lesions coupled with a pull-down assay and followed by next-generation sequencing, this method is easy to perform and can produce compelling results with minimal coverage. RADD-seq enables the localization of both DNA damage and repair sites for a wide range of single-strand damage types. Using this technique, we created a genome-wide map of the oxidation DNA damage lesion 8-oxo-7,8-dihydroguanine before and after repair. Oxidation lesions were heterogeneously distributed along the human genome, with less damage occurring in tight chromatin regions. Furthermore, we showed repair is prioritized for highly expressed, essential genes and in open chromatin regions. RADD-seq sheds light on cellular repair mechanisms and is capable of identifying genomic hotspots prone to mutation.

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“…Cellular DNA damage lesions are continuously induced by exposure to various exogenous and endogenous agents, and are a major cause for genomic instability. A labeling scheme called RADD (repair-assisted damage detection), was developed to fluorescently label single-strand lesions in vitro by a two-step process [107][108][109]. First, native repair enzymes excise the damaged bases and leave a single-strand gap at the damage locus.…”

Section: Optical Mapping Of Epigeneticsmentioning

confidence: 99%

Single-molecule optical genome mapping in nanochannels: multidisciplinarity at the nanoscale

Jeffet

Margalit

Michaeli

et al. 2021

Essays in Biochemistry

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The human genome contains multiple layers of information that extend beyond the genetic sequence. In fact, identical genetics do not necessarily yield identical phenotypes as evident for the case of two different cell types in the human body. The great variation in structure and function displayed by cells with identical genetic background is attributed to additional genomic information content. This includes large-scale genetic aberrations, as well as diverse epigenetic patterns that are crucial for regulating specific cell functions. These genetic and epigenetic patterns operate in concert in order to maintain specific cellular functions in health and disease. Single-molecule optical genome mapping is a high-throughput genome analysis method that is based on imaging long chromosomal fragments stretched in nanochannel arrays. The access to long DNA molecules coupled with fluorescent tagging of various genomic information presents a unique opportunity to study genetic and epigenetic patterns in the genome at a single-molecule level over large genomic distances. Optical mapping entwines synergistically chemical, physical, and computational advancements, to uncover invaluable biological insights, inaccessible by sequencing technologies. Here we describe the method’s basic principles of operation, and review the various available mechanisms to fluorescently tag genomic information. We present some of the recent biological and clinical impact enabled by optical mapping and present recent approaches for increasing the method’s resolution and accuracy. Finally, we discuss how multiple layers of genomic information may be mapped simultaneously on the same DNA molecule, thus paving the way for characterizing multiple genomic observables on individual DNA molecules.

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Rapid Quantification of Oxidation and UV Induced DNA Damage by Repair Assisted Damage Detection-(Rapid RADD)

Cited by 12 publications

References 31 publications

Genome wide mapping of DNA lesions by Repair Assisted Damage Detection sequencing – RADD-Seq

Genome wide mapping of DNA lesions by Repair Assisted Damage Detection sequencing – RADD-Seq

From single-molecule to genome-wide mapping of DNA lesions: repair-assisted damage detection sequencing

Single-molecule optical genome mapping in nanochannels: multidisciplinarity at the nanoscale

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