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

ABSTRACT: 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.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE62181 | GEO | 2015/01/26

SECONDARY ACCESSION(S): PRJNA263378

REPOSITORIES: GEO

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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

Evidence that DNA polymerase δ contributes to initiation of leading strand DNA replication in Saccharomyces cerevisiae

Project description:To investigate nuclear DNA replication enzymology in vivo, we have studied Saccharomyces cerevisiae strains containing a pol2-16 mutation that inactivates the catalytic activities of DNA polymerase δ (Pol δ). Although pol2-16 mutants survive, their spore colonies are very tiny, with increased doubling time, larger than normal cells, aberrant nuclei, and rapid suppressor mutation accumulation. These phenotypes reveal a severe growth defect that is distinct from that of strains that lack Pol δ proofreading (pol2-4), consistent with the idea that Pol δ is the major leading strand replicase. Ribonucleotides are also incorporated into the pol2-16 genome in patterns consistent with leading strand replication by Pol δ when Pol δ is absent. More importantly, ribonucleotide distributions at replication origins suggest that in strains encoding all three replicases, Pol δ contributes to initiation of leading-strand replication. We describe two possible models.

2018-02-27 | GSE101698 | 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

Genome-wide tracking of Escherichia coli DNA polymerase V to the lagging strand during the SOS response

Project description:Ribonucleotides are frequently incorporated into DNA and can be used as a marker of DNA replication enzymology. To investigate on a genome-wide scale, how E. coli pol V accesses undamaged chromosomal DNA during the SOS response, we mapped the location of ribonucleotides incorporated by steric gate variants of pol V across the entire E. coli genome. To do so, we used strains that are deficient in ribonucleotide excision repair (DrnhB), constitutively express all SOS-regulated genes [lexA(Def)] and constitutively “activated” RecA* (recA730). The strains also harbor two steric gate variants of E. coli pol V (Y11A or F10L), or a homolog of pol V, (pol VR391-Y13A). Ribonucleotides are frequently incorporated by the pol V-Y11A and pol VR391-Y13A variants, with a preference to the lagging strand. In contrast, the pol V-F10L variant incorporates less ribonucleotides and no strand preference was observed. Sharp transitions in strand specificity are observed at replication origin (oriC), while a gradient is observed at the termination region. To activate RecA* in a recA+ strain, we treated the strains with ciprofloxacin and genome-wide mapped the location of the incorporated ribonucleotides. Again, the polV-Y11A steric gate variant, exhibited a lagging strand preference. Our data is consistent with a specific role for pol V in lagging strand DNA synthesis across the entire E. coli genome during the SOS response.

2021-03-22 | GSE158570 | GEO

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

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

2015-01-26 | GSE64521 | 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

Evaluation of Repair Activity by Quantification of Ribonucleotides in the Genome

Project description:Ribonucleotides incorporated in the genome are a source of endogenous DNA damage, and also serve as signals for repair. Although recent advances of ribonucleotide detection by sequencing, the balance between incorporation and repair of ribonucleotides has not been elucidated. Here, we describe a competitive sequencing method, Ribonucleotide Scanning Quantification sequencing (RiSQ-seq), which enables absolute quantification of misincorporated ribonucleotides throughout the genome by background normalization and standard adjustment within a single sample. RiSQ-seq analysis of cells harboring wild-type DNA polymerases revealed that ribonucleotides were incorporated non-uniformly in the genome with a 3’-shifted distribution and preference for GC sequences. Although ribonucleotide profiles in wild-type and repair-deficient mutant strains showed a similar pattern, direct comparison of distinct ribonucleotide levels in the strains by RiSQ-seq enabled evaluation of ribonucleotide excision repair activity at base resolution and revealed the strand bias of repair. The distinct preferences of ribonucleotide incorporation and repair create vulnerable regions associated with indel hotspots, suggesting that repair at sites of ribonucleotide misincorporation serves to maintain genome integrity and that RiSQ-seq can provide an estimate of indel risk.

2021-05-21 | GSE85130 | GEO

Ribonucleotide excision repair is essential to prevent skin cancer [CD49f+ epidermal cells]

Project description:Large numbers of ribonucleotides are incorporated into the eukaryotic nuclear genome during S-phase due to imperfect discrimination against ribonucleoside triphosphates by the replicative DNA polymerases. Ribonucleotides, by far the most common DNA lesion in replicating cells, destabilize the DNA, and an evolutionarily conserved DNA repair machinery, ribonucleotide excision repair (RER), ensures ribonucleotide removal. Complete lack of RER is embryonically lethal. Partial loss-of-function mutations in the genes encoding subunits of RNase H2, the enzyme essential for initiation of RER, cause the SLE-related type I interferonopathy Aicardi-Goutières syndrome. Here we establish that selective inactivation of RER in mouse epidermis results in spontaneous DNA damage, epidermal hyperproliferation associated with loss of hair follicle stem cells and hair follicle function. The animals develop keratinocyte intraepithelial neoplasia and invasive squamous cell carcinoma with complete penetrance, despite potent type I interferon production and skin inflammation. Compromised RER-mediated genome maintenance might represent an important tumor-promoting principle in human cancer.

2019-05-29 | GSE115003 | GEO

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