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Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA.

ABSTRACT: Abundant ribonucleotide incorporation in DNA during replication and repair has profound consequences for genome stability, but the global distribution of ribonucleotide incorporation is unknown. We developed ribose-seq, a method for capturing unique products generated by alkaline cleavage of DNA at embedded ribonucleotides. High-throughput sequencing of these fragments in DNA from the yeast Saccharomyces cerevisiae revealed widespread ribonucleotide distribution, with a strong preference for cytidine and guanosine, and identified hotspots of ribonucleotide incorporation in nuclear and mitochondrial DNA. Ribonucleotides were primarily incorporated on the newly synthesized leading strand of nuclear DNA and were present upstream of (G+C)-rich tracts in the mitochondrial genome. Ribose-seq is a powerful tool for the systematic profiling of ribonucleotide incorporation in genomic DNA.

SUBMITTER: Koh KD

PROVIDER: S-EPMC4686381 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA.

Koh Kyung Duk KD Balachander Sathya S Hesselberth Jay R JR Storici Francesca F

Nature methods 20150126 3

Abundant ribonucleotide incorporation in DNA during replication and repair has profound consequences for genome stability, but the global distribution of ribonucleotide incorporation is unknown. We developed ribose-seq, a method for capturing unique products generated by alkaline cleavage of DNA at embedded ribonucleotides. High-throughput sequencing of these fragments in DNA from the yeast Saccharomyces cerevisiae revealed widespread ribonucleotide distribution, with a strong preference for cyt ...[more]

PMID: 25622106

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Addendum: Ribose-seq: global mapping of ribonucleotides embedded in genomic DNA.

Project description: Not available

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Mapping ribonucleotides embedded in genomic DNA to single-nucleotide resolution using Ribose-Map.

Project description:The most common nonstandard nucleotides found in genomic DNA are ribonucleotides. Although ribonucleotides are frequently incorporated into DNA by replicative DNA polymerases, very little is known about the distribution and signatures of ribonucleotides incorporated into DNA. Recent advances in high-throughput ribonucleotide sequencing can capture the exact locations of ribonucleotides in genomic DNA. Ribose-Map is a user-friendly, standardized bioinformatics toolkit for the comprehensive analysis of ribonucleotide sequencing experiments. It allows researchers to map the locations of ribonucleotides in DNA to single-nucleotide resolution and identify biological signatures of ribonucleotide incorporation. In addition, it can be applied to data generated using any currently available high-throughput ribonucleotide sequencing technique, thus standardizing the analysis of ribonucleotide sequencing experiments and allowing direct comparisons of results. This protocol describes in detail how to use Ribose-Map to analyze ribonucleotide sequencing data, including preparing the reads for analysis, locating the genomic coordinates of ribonucleotides, exploring the genome-wide distribution of ribonucleotides, determining the nucleotide sequence context of ribonucleotides and identifying hotspots of ribonucleotide incorporation. Ribose-Map does not require background knowledge of ribonucleotide sequencing analysis and assumes only basic command-line skills. The protocol requires less than 3 h of computing time for most datasets and ~30 min of hands-on time. Ribose-Map is available at https://github.com/agombolay/ribose-map .

| S-EPMC9444191 | biostudies-literature

Ribose-Map: a bioinformatics toolkit to map ribonucleotides embedded in genomic DNA.

Project description:Recent advances in high-throughput sequencing techniques have made it possible to tag ribonucleoside monophosphates (rNMPs) embedded in genomic DNA for sequencing. rNMP sequencing experiments generate large, complex datasets that require efficient, scalable software that can accurately map embedded rNMPs independently of the particular sequencing technique used. Current computational pipelines designed to map rNMPs embedded in genomic DNA are customized for data generated using only one type of rNMP sequencing technique. To standardize the processing and analysis of rNMP sequencing experiments, we developed Ribose-Map. Through a series of analytical modules, Ribose-Map transforms raw sequencing data into summary datasets and publication-ready visualizations of results, allowing biologists to identify sites of embedded rNMPs, study the nucleotide sequence context of these rNMPs and explore their genome-wide distribution. By accommodating data from any of the available rNMP sequencing techniques, Ribose-Map can increase the reproducibility of rNMP sequencing experiments and enable a head-to-head comparison of these experiments.

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Global mapping of ribonucleotides embedded in DNA

Project description:Capture and massively parallel DNA sequencing of ribonucleotides embedded in S. cerevisiae genomic DNA

2015-01-15 | GSE61464 | GEO

Global mapping of ribonucleotides embedded in DNA

Project description:Capture and massively parallel DNA sequencing of ribonucleotides embedded in S. cerevisiae genomic DNA We developed a new method to map the positions of ribonucleotides embedded in DNA using the unique specificity of A. thaliana tRNA ligase. Ribonucleotides were generated in budding yeasts of different genetic backgrounds and mapped to single nucleotide resolution using the new method.

2015-01-15 | E-GEOD-61464 | biostudies-arrayexpress

Ribonucleotides embedded in genomic DNA of Aicardi-Goutières syndrome (AGS)-orthologous mutants of Saccharomyces cerevisiae using ribose-seq protocol

Project description:Ribonucleoside monophosphates (rNMPs) are abundantly found in DNA of cells, with over a million counts in the mammalian genome and thousands in budding yeast DNA. The embedded rNMPs alter DNA properties, increasing DNA fragility and mutability. Mutations in any of the three subunits of ribonuclease (RNase) H2, a key enzyme for rNMP removal, are associated with the Aicardi-Goutières syndrome (AGS), a severe neurological disorder. Here, we engineered two AGS orthologous mutations in Saccharomyces cerevisiae: rnh201-G42S and rnh203-K46W. Using the ribose-seq technique and the Ribose-Map bioinformatics tool, we studied the rNMP incorporation frequency, distribution, composition, and patterns in these yeast mutants. We found significantly more abundant rNMPs in the nuclear genome of rnh201-G42S than in wild-type and rnh203-K46W-mutant cells. Moreover, we observed distinct sequence contexts and genomic sites with abundant rNMP incorporation (hotspots) in rnh201-G42S and rnh203-K46W cells, anticipating similar rNMP patterns in human DNA carrying the orthologous AGS mutations.

2023-12-25 | GSE240399 | GEO

Ribonucleotides embedded in template DNA impair mitochondrial RNA polymerase progression.

Project description:Human mitochondria lack ribonucleotide excision repair pathways, causing misincorporated ribonucleotides (rNMPs) to remain embedded in the mitochondrial genome. Previous studies have demonstrated that human mitochondrial DNA polymerase γ can bypass a single rNMP, but that longer stretches of rNMPs present an obstacle to mitochondrial DNA replication. Whether embedded rNMPs also affect mitochondrial transcription has not been addressed. Here we demonstrate that mitochondrial RNA polymerase elongation activity is affected by a single, embedded rNMP in the template strand. The effect is aggravated at stretches with two or more consecutive rNMPs in a row and cannot be overcome by addition of the mitochondrial transcription elongation factor TEFM. Our findings lead us to suggest that impaired transcription may be of functional relevance in genetic disorders associated with imbalanced nucleotide pools and higher levels of embedded rNMPs.

| S-EPMC8789056 | biostudies-literature

Unlike the <i>Escherichia coli</i> counterpart, archaeal RNase HII cannot process ribose monophosphate abasic sites and oxidized ribonucleotides embedded in DNA.

Project description:The presence of ribonucleoside monophosphates (rNMPs) in nuclear DNA decreases genome stability. To ensure survival despite rNMP insertions, cells have evolved a complex network of DNA repair mechanisms, in which the ribonucleotide excision repair pathway, initiated by type 2 RNase H (RNase HII/2), plays a major role. We recently demonstrated that eukaryotic RNase H2 cannot repair damage, that is, ribose monophosphate abasic (both apurinic or apyrimidinic) site (rAP) or oxidized rNMP embedded in DNA. Currently, it remains unclear why RNase H2 is unable to repair these modified nucleic acids having either only a sugar moiety or an oxidized base. Here, we compared the endoribonuclease specificity of the RNase HII enzymes from the archaeon Pyrococcus abyssi and the bacterium Escherichia coli, examining their ability to process damaged rNMPs embedded in DNA in vitro We found that E. coli RNase HII cleaves both rAP and oxidized rNMP sites. In contrast, like the eukaryotic RNase H2, P. abyssi RNase HII did not display any rAP or oxidized rNMP incision activities, even though it recognized them. Notably, the archaeal enzyme was also inactive on a mismatched rNMP, whereas the E. coli enzyme displayed a strong preference for the mispaired rNMP over the paired rNMP in DNA. On the basis of our biochemical findings and also structural modeling analyses of RNase HII/2 proteins from organisms belonging to all three domains of life, we propose that RNases HII/2's dual roles in ribonucleotide excision repair and RNA/DNA hydrolysis result in limited acceptance of modified rNMPs embedded in DNA.

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Ribonucleotides embedded in genomic DNA of Aicardi-Goutières syndrome (AGS)-orthologous mutants of Saccharomyces cerevisiae using ribose-seq protocol

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Precision digital mapping of endogenous and induced genomic DNA breaks by INDUCE-seq.

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