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Genome wide maps of replicative polymerase contributions to the fully replicated yeast genome

ABSTRACT: We report the application of high through-put tag sequencing to measure the location and strand of DNA embedded ribonucleotides in the yeast genome. Mutations in the catalytic subunits of the polymerases (pol1-L868M, pol2-M644G and pol3-L612M) lead to the increased incorporation of ribonucleotides during DNA replication, providing an in vivo label with which to track the contribution of each polymerase to the fully replicated genome. Yeast strains used in this study are deleted for rnh201, encoding the catalytic subunit of the RNase H2 gene so that embedded ribonucleotides are not rapidly removed by ribonucleotide excision repair following DNA replication. Analysis of this data demonstrates that polymerase alpha contributes to the fully replicated genome.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE64521 | GEO | 2015/01/26

SECONDARY ACCESSION(S): PRJNA271170

REPOSITORIES: GEO

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Genome wide maps of replicative polymerase contributions to the fully replicated yeast genome

Project description:We report the application of high through-put tag sequencing to measure the location and strand of DNA embedded ribonucleotides in the yeast genome. Mutations in the catalytic subunits of the polymerases (pol1-L868M, pol2-M644G and pol3-L612M) lead to the increased incorporation of ribonucleotides during DNA replication, providing an in vivo label with which to track the contribution of each polymerase to the fully replicated genome. Yeast strains used in this study are deleted for rnh201, encoding the catalytic subunit of the RNase H2 gene so that embedded ribonucleotides are not rapidly removed by ribonucleotide excision repair following DNA replication. Analysis of this data demonstrates that polymerase alpha contributes to the fully replicated genome. Sequencing of DNA embedded ribonucleotides in S. cerevisiae strains to map the contribution of replicative polymerases to the fully replicated genome.

2015-01-26 | E-GEOD-64521 | biostudies-arrayexpress

Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation

Project description:Multiple DNA polymerases are needed to replicate genetic information. Here we describe the use of ribonucleotide incorporation as a biomarker of replication enzymology in vivo. We find that ribonucleotides are incorporated into the yeast nuclear genome in replicase specific and strand-specific patterns that identify replication origins and where polymerase switching occurs. Ribonucleotide density varies across the genome as a function of the replicase, base, local sequence and proximity to nucleosomes and transcription start sites. Ribonucleotides are present in one strand at high densityat mitochondrial replication origins, implying unidirectional replication of a circular genome. The evolutionary conservation of the enzymes that incorporate and process ribonucleotides in DNA suggests that the use of ribonucleotides as biomarkers of DNA synthesis in cells will have widespread applicability. Mapping genomic ribonucleotides in 14 Saccharomyces cerevisiae strains (seven DNA polymerase backgrounds, with or without RNH201), via HydEn-seq (end sequencing of genomic fragments generated by alkaline hydrolysis).

2015-01-26 | E-GEOD-62181 | biostudies-arrayexpress

Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA

Project description:Previous work has demonstrated the presence of ribonucleotides in human mitochondrial DNA (mtDNA) and in the present study we use a genome-wide approach to precisely map the location of these. We find that ribonucleotides are distributed evenly between the heavy- and light-strand of mtDNA. The relative levels of incorporated ribonucleotides reflect that DNA polymerase γ discriminates the four ribonucleotides differentially during DNA synthesis. The observed pattern is also dependent on the mitochondrial deoxyribonucleotide (dNTP) pools and disease-causing mutations that change these pools alter both the absolute and relative levels of incorporated ribonucleotides. Our analyses strongly suggest that DNA polymerase γ-dependent incorporation is the main source of ribonucleotides in mtDNA and argues against the existence of a mitochondrial ribonucleotide excision repair pathway in human cells. Furthermore, we clearly demonstrate that when dNTP pools are limiting, ribonucleotides serve as a source of building blocks to maintain DNA replication and genome stability. Increased levels of embedded ribonucleotides in patient cells with disturbed nucleotide pools may constitute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication

2017-02-13 | GSE90054 | GEO

Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation

2015-01-26 | GSE62181 | GEO

Saccharomyces cerevisiae

Project description:Genome wide maps of replicative polymerase contributions to the fully replicated yeast genome

| PRJNA271170 | ENA

The Asymmetric Distribution of RNAPII and Nucleosomes on Replicated Daughter Genomes is caused by Differences in Replication Timing between the Lagging and the Leading Strand [ChIP]

Project description:Chromatin features are thought to have a role in the epigenetic transmission of transcription states from one cell generation to the next. It is unclear how chromatin structure survives disruptions caused by genomic replication or if chromatin features are instructive of the transcription state of the underlying gene. We developed a method to monitor budding yeast replication, transcription and chromatin maturation dynamics on each daughter genome in parallel, with which we identified clusters of secondary origins surrounding known origins. We find a difference in the timing of lagging and leading strand replication on the order of minutes at most yeast genes. We propose a model in which the majority of old histones and RNAPII bind to the gene copy that replicated first, while newly synthesized nucleosomes are assembled on the copy that replicated second. RNAPII enrichment then shifts to the sister copy that replicated second. The order of replication is largely determined by genic orientation: if transcription and replication are co-directional the leading strand replicates first; if they are counter-directional the lagging strand replicates first. A mutation in the Mcm2 subunit of the replicative helicase Mcm2-7 which impairs Mcm2 interactions with histone H3 slows down replication forks but does not qualitatively change the asymmetry in nucleosome distribution observed in the WT. Active transcription states are inherited simultaneously and independently of their underlying chromatin states through the recycling of the transcription machinery and old histones, respectively. Transcription thus actively contributes to the reestablishment of the active chromatin state.

2021-12-18 | GSE160507 | GEO

The Asymmetric Distribution of RNAPII and Nucleosomes on Replicated Daughter Genomes is caused by Differences in Replication Timing between the Lagging and the Leading Strand [RNA-seq]

2021-12-18 | GSE160265 | GEO

The Asymmetric Distribution of RNAPII and Nucleosomes on Replicated Daughter Genomes is caused by Differences in Replication Timing between the Lagging and the Leading Strand [ChIP-seq]

2021-12-18 | GSE160262 | GEO

Aberrant ribonucleotide incorporation and multiple deletions in mitochondrial DNA of the murine MPV17 disease model [EmRibo-seq]

Project description:All DNA polymerases misincorporate ribonucleotides despite their preference for deoxyribonucleotides, and analysis of cultured cells indicates that mammalian mitochondrial DNA (mtDNA) tolerates such replication errors. However, it is not clear to what extent ribonucleotides are incorporated into the mtDNA of solid tissues, or whether they might play a role in human pathologies. Here, we show the DNA in mitochondria of solid tissues contains many more embedded ribonucleotides than that of cultured cells, consistent with the former’s high ratio of ribonucleotide to deoxynucleotide triphosphates and that rAMPs are the predominant ribosubstitution events. This pattern changes in a mouse model of Mpv17 deficiency, as rGMPs are the major embedded ribonucleotides of mtDNA. However, while mitochondrial dGTP is reduced in the liver of the KO mice, the brain shows no change in the overall dGTP pool, leading us to infer that Mpv17 determines the local concentration or quality of dGTP. Embedded rGMPs are expected to impede DNA replication more than other rNMPs, and elevated rGMP incorporation is associated with early-onset mtDNA depletion in liver and late-onset multiple deletions in brain of the Mpv17 ablated mice. These findings suggest that aberrant ribonucleotide incorporation is a primary mtDNA abnormality that can result in pathology.

2017-10-23 | GSE103429 | GEO

Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired

Project description:Misincorporation of ribonucleotides into DNA during genome replication has recently become recognized as a significant source of genomic instability. The frequency of ribonucleotides in the genome is determined by dNTP/rNTP ratios, the ability of the DNA polymerases to discriminate against ribonucleotides, and by the capacity of repair mechanisms to remove misincorporated ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove ribonucleotides, we challenged these processes by imbalancing cellular dNTP pools. Using a collection of yeast strains with altered dNTP pools, we discovered an inverse relationship between the concentration of individual dNTPs and the amount of the corresponding ribonucleotides incorporated in mitochondrial DNA, while in nuclear DNA the ribonucleotide pattern was only altered in the absence of ribonucleotide excision repair. Our analysis uncovers major differences in ribonucleotide repair between the two genomes and provides concrete evidence that yeast mitochondria lack mechanisms for repair of misincorporated ribonucleotides.

2017-11-07 | GSE100352 | GEO

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