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Thomas A. Kunkel - One of the best experts on this subject based on the ideXlab platform.
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Ribonucleotide incorporation into DNA during DNA replication and its consequences.
Critical reviews in biochemistry and molecular biology, 2021Co-Authors: Zhi-xiong Zhou, Scott A Lujan, Jessica S Williams, Thomas A. KunkelAbstract:Ribonucleotides are the most abundant non-canonical nucleotides in the genome. Their vast presence and influence over genome biology is becoming increasingly appreciated. Here we review the recent progress made in understanding their genomic presence, incorporation characteristics and usefulness as biomarkers for polymerase enzymology. We also discuss ribonucleotide processing, the genetic consequences of unrepaired Ribonucleotides in DNA and evidence supporting the significance of their transient presence in the nuclear genome.
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genome wide mutagenesis resulting from topoisomerase 1 processing of unrepaired Ribonucleotides in dna
DNA Repair, 2019Co-Authors: Jessica S Williams, Scott A Lujan, Alan B Clark, Zhi-xiong Zhou, Adam B Burkholder, David C Fargo, Thomas A. KunkelAbstract:Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2-5 base pair deletions (Δ2-5bp) in repetitive DNA are a signature of unrepaired Ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol e, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.
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ribonucleotide excision repair is essential to prevent squamous cell carcinoma of the skin
Cancer Research, 2018Co-Authors: Bjorn Hiller, Anja Hoppe, Christa Haase, Christina Hiller, Nadja Schubert, Werner Muller, Martin A M Reijns, Andrew P Jackson, Thomas A. KunkelAbstract:Because of imperfect discrimination against ribonucleoside triphosphates by the replicative DNA polymerases, large numbers of Ribonucleotides are incorporated into the eukaryotic nuclear genome during S-phase. 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. Whereas 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-Goutieres syndrome. Here, we demonstrate that selective inactivation of RER in mouse epidermis results in spontaneous DNA damage and epidermal hyperproliferation associated with loss of hair follicle stem cells and hair follicle function. The animals developed keratinocyte intraepithelial neoplasia and invasive squamous cell carcinoma with complete penetrance, despite potent type I interferon production and skin inflammation. These results suggest that compromises to RER-mediated genome maintenance might represent an important tumor-promoting principle in human cancer.Significance: Selective inactivation of ribonucleotide excision repair by loss of RNase H2 in the murine epidermis results in spontaneous DNA damage, type I interferon response, skin inflammation, and development of squamous cell carcinoma. Cancer Res; 78(20); 5917-26. ©2018 AACR.
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Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis.
Journal of visualized experiments : JoVE, 2018Co-Authors: Zhi-xiong Zhou, Jessica S Williams, Thomas A. KunkelAbstract:The presence of Ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the Ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of Ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.
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mapping Ribonucleotides incorporated into dna by hyd rolytic en d seq uencing
Methods of Molecular Biology, 2018Co-Authors: Clinton D Orebaugh, Anders R Clausen, Scott A Lujan, Adam B Burkholder, Thomas A. KunkelAbstract:Ribonucleotides embedded within DNA render the DNA sensitive to the formation of single-stranded breaks under alkali conditions. Here, we describe a next-generation sequencing method called hydrolytic end sequencing (HydEn-seq) to map Ribonucleotides inserted into the genome of Saccharomyce cerevisiae strains deficient in ribonucleotide excision repair. We use this method to map several genomic features in wild-type and replicase variant yeast strains.
Jessica S Williams - One of the best experts on this subject based on the ideXlab platform.
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Ribonucleotide incorporation into DNA during DNA replication and its consequences.
Critical reviews in biochemistry and molecular biology, 2021Co-Authors: Zhi-xiong Zhou, Scott A Lujan, Jessica S Williams, Thomas A. KunkelAbstract:Ribonucleotides are the most abundant non-canonical nucleotides in the genome. Their vast presence and influence over genome biology is becoming increasingly appreciated. Here we review the recent progress made in understanding their genomic presence, incorporation characteristics and usefulness as biomarkers for polymerase enzymology. We also discuss ribonucleotide processing, the genetic consequences of unrepaired Ribonucleotides in DNA and evidence supporting the significance of their transient presence in the nuclear genome.
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genome wide mutagenesis resulting from topoisomerase 1 processing of unrepaired Ribonucleotides in dna
DNA Repair, 2019Co-Authors: Jessica S Williams, Scott A Lujan, Alan B Clark, Zhi-xiong Zhou, Adam B Burkholder, David C Fargo, Thomas A. KunkelAbstract:Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2-5 base pair deletions (Δ2-5bp) in repetitive DNA are a signature of unrepaired Ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol e, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.
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Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis.
Journal of visualized experiments : JoVE, 2018Co-Authors: Zhi-xiong Zhou, Jessica S Williams, Thomas A. KunkelAbstract:The presence of Ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the Ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of Ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.
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Studying Topoisomerase 1-Mediated Damage at Genomic Ribonucleotides.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Jessica S Williams, Thomas A. KunkelAbstract:Ribonucleotides incorporated into DNA by the DNA polymerases can be incised by Topoisomerase 1 (Top1) to initiate removal of Ribonucleotides from the genome. This Top1-dependent ribonucleotide removal has been demonstrated to result in multiple forms of genome instability in yeast. Here, we describe both quantitative and qualitative assays to identify mutations and other forms of DNA damage resulting from Top1-cleavage at unrepaired genomic Ribonucleotides.
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the role of rnase h2 in processing Ribonucleotides incorporated during dna replication
DNA Repair, 2017Co-Authors: Jessica S Williams, Daniel B Gehle, Thomas A. KunkelAbstract:Abstract Saccharomyces cerevisiae RNase H2 resolves RNA-DNA hybrids formed during transcription and it incises DNA at single Ribonucleotides incorporated during nuclear DNA replication. To distinguish between the roles of these two activities in maintenance of genome stability, here we investigate the phenotypes of a mutant of yeast RNase H2 ( rnh201-RED ; ribonucleotide excision defective) that retains activity on RNA-DNA hybrids but is unable to cleave single Ribonucleotides that are stably incorporated into the genome. The rnh201-RED mutant was expressed in wild type yeast or in a strain that also encodes a mutant allele of DNA polymerase e ( pol2-M644G ) that enhances ribonucleotide incorporation during DNA replication. Similar to a strain that completely lacks RNase H2 ( rnh201 Δ), the pol2-M644G rnh201-RED strain exhibits replication stress and checkpoint activation. Moreover, like its null mutant counterpart, the double mutant pol2-M644G rnh201-RED strain and the single mutant rnh201-RED strain delete 2–5 base pairs in repetitive sequences at a high rate that is topoisomerase 1-dependent. The results highlight an important role for RNase H2 in maintaining genome integrity by removing single Ribonucleotides incorporated during DNA replication.
Anders R Clausen - One of the best experts on this subject based on the ideXlab platform.
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mapping Ribonucleotides incorporated into dna by hyd rolytic en d seq uencing
Methods of Molecular Biology, 2018Co-Authors: Clinton D Orebaugh, Anders R Clausen, Scott A Lujan, Adam B Burkholder, Thomas A. KunkelAbstract:Ribonucleotides embedded within DNA render the DNA sensitive to the formation of single-stranded breaks under alkali conditions. Here, we describe a next-generation sequencing method called hydrolytic end sequencing (HydEn-seq) to map Ribonucleotides inserted into the genome of Saccharomyce cerevisiae strains deficient in ribonucleotide excision repair. We use this method to map several genomic features in wild-type and replicase variant yeast strains.
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Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Paulina H. Wanrooij, Anders R Clausen, Martin K. M. Engqvist, Josefin M. E. Forslund, Clara Navarrete, Anna Karin Nilsson, Juhan Sedman, Sjoerd Wanrooij, Andrei ChabesAbstract:Incorporation of Ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of Ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against Ribonucleotides, and by the capacity of repair mechanisms to remove incorporated Ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove Ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. 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 removal of Ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange.
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Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA.
PLoS genetics, 2017Co-Authors: Anna-karin Berglund, Martin K. M. Engqvist, Clara Navarrete, Emily Hoberg, Zsolt Szilagyi, Robert W. Taylor, Claes M. Gustafsson, Maria Falkenberg, Anders R ClausenAbstract: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. Increased levels of embedded Ribonucleotides in patient cells with disturbed nucleotide pools may contribute to a pathogenic mechanism that affects mtDNA stability and impair new rounds of mtDNA replication.
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Evidence that processing of Ribonucleotides in DNA by topoisomerase 1 is leading-strand specific
Nature Structural & Molecular Biology, 2015Co-Authors: Jessica S Williams, Anders R Clausen, Scott A Lujan, Alan B Clark, Andrei Chabes, Lisette Marjavaara, Peter M Burgers, Thomas A. KunkelAbstract:Ribonucleotides incorporated during DNA replication are removed by RNase H2–dependent ribonucleotide excision repair (RER). In RER-defective yeast, topoisomerase 1 (Top1) incises DNA at unrepaired Ribonucleotides, initiating their removal, but this is accompanied by RNA-DNA–damage phenotypes. Here we show that these phenotypes are incurred by a high level of Ribonucleotides incorporated by a leading strand–replicase variant, DNA polymerase (Pol) ɛ, but not by orthologous variants of the lagging-strand replicases, Pols α or δ. Moreover, loss of both RNases H1 and H2 is lethal in combination with increased ribonucleotide incorporation by Pol ɛ but not by Pols α or δ. Several explanations for this asymmetry are considered, including the idea that Top1 incision at Ribonucleotides relieves torsional stress in the nascent leading strand but not in the nascent lagging strand, in which preexisting nicks prevent the accumulation of superhelical tension. In the absence of RNase H2, Ribonucleotides incorporated during DNA replication can be processed by Top1. This activity is directed to the nascent leading strand, because gaps in the lagging strand would limit torsional tension.
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evidence that processing of Ribonucleotides in dna by topoisomerase 1 is leading strand specific
Nature Structural & Molecular Biology, 2015Co-Authors: Jessica S Williams, Anders R Clausen, Scott A Lujan, Alan B Clark, Andrei Chabes, Lisette Marjavaara, Peter M. J. Burgers, Thomas A. KunkelAbstract:Ribonucleotides incorporated during DNA replication are removed by RNase H2-dependent ribonucleotide excision repair (RER). In RER-defective yeast, topoisomerase 1 (Top1) incises DNA at unrepaired ...
Scott A Lujan - One of the best experts on this subject based on the ideXlab platform.
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Ribonucleotide incorporation into DNA during DNA replication and its consequences.
Critical reviews in biochemistry and molecular biology, 2021Co-Authors: Zhi-xiong Zhou, Scott A Lujan, Jessica S Williams, Thomas A. KunkelAbstract:Ribonucleotides are the most abundant non-canonical nucleotides in the genome. Their vast presence and influence over genome biology is becoming increasingly appreciated. Here we review the recent progress made in understanding their genomic presence, incorporation characteristics and usefulness as biomarkers for polymerase enzymology. We also discuss ribonucleotide processing, the genetic consequences of unrepaired Ribonucleotides in DNA and evidence supporting the significance of their transient presence in the nuclear genome.
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genome wide mutagenesis resulting from topoisomerase 1 processing of unrepaired Ribonucleotides in dna
DNA Repair, 2019Co-Authors: Jessica S Williams, Scott A Lujan, Alan B Clark, Zhi-xiong Zhou, Adam B Burkholder, David C Fargo, Thomas A. KunkelAbstract:Ribonucleotides are the most common non-canonical nucleotides incorporated into DNA during replication, and their processing leads to mutations and genome instability. Yeast mutation reporter systems demonstrate that 2-5 base pair deletions (Δ2-5bp) in repetitive DNA are a signature of unrepaired Ribonucleotides, and that these events are initiated by topoisomerase 1 (Top1) cleavage. However, a detailed understanding of the frequency and locations of ribonucleotide-dependent mutational events across the genome has been lacking. Here we present the results of genome-wide mutational analysis of yeast strains deficient in Ribonucleotide Excision Repair (RER). We identified mutations that accumulated over thousands of generations in strains expressing either wild-type or variant replicase alleles (M644G Pol e, L612M Pol δ, L868M Pol α) that confer increased ribonucleotide incorporation into DNA. Using a custom-designed mutation-calling pipeline called muver (for mutationes verificatae), we observe a number of surprising mutagenic features. This includes a 24-fold preferential elevation of AG and AC relative to AT dinucleotide deletions in the absence of RER, suggesting specificity for Top1-initiated deletion mutagenesis. Moreover, deletion rates in di- and trinucleotide repeat tracts increase exponentially with tract length. Consistent with biochemical and reporter gene mutational analysis, these deletions are no longer observed upon deletion of TOP1. Taken together, results from these analyses demonstrate the global impact of genomic ribonucleotide processing by Top1 on genome integrity.
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mapping Ribonucleotides incorporated into dna by hyd rolytic en d seq uencing
Methods of Molecular Biology, 2018Co-Authors: Clinton D Orebaugh, Anders R Clausen, Scott A Lujan, Adam B Burkholder, Thomas A. KunkelAbstract:Ribonucleotides embedded within DNA render the DNA sensitive to the formation of single-stranded breaks under alkali conditions. Here, we describe a next-generation sequencing method called hydrolytic end sequencing (HydEn-seq) to map Ribonucleotides inserted into the genome of Saccharomyce cerevisiae strains deficient in ribonucleotide excision repair. We use this method to map several genomic features in wild-type and replicase variant yeast strains.
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Processing Ribonucleotides incorporated during eukaryotic DNA replication
Nature Reviews Molecular Cell Biology, 2016Co-Authors: Jessica S Williams, Scott A Lujan, Thomas A. KunkelAbstract:The information encoded in DNA is influenced by the presence of non-canonical nucleotides, the most frequent of which are Ribonucleotides. In this Review, we discuss recent discoveries about ribonucleotide incorporation into DNA during replication by the three major eukaryotic replicases, DNA polymerases α, δ and ε. The presence of Ribonucleotides in DNA causes short deletion mutations and may result in the generation of single- and double-strand DNA breaks, leading to genome instability. We describe how these Ribonucleotides are removed from DNA through ribonucleotide excision repair and by topoisomerase I. We discuss the biological consequences and the physiological roles of Ribonucleotides in DNA, and consider how deficiencies in their removal from DNA may be important in the aetiology of disease. Ribonucleotides are incorporated into DNA during replication. Ribonucleotides can be removed during ribonucleotide excision repair or topoisomerase I-initiated processing. Failure of ribonucleotide removal is associated with genome instability in the form of mutagenesis, replication stress, DNA breaks and chromosomal rearrangements. Human diseases, including autoimmune disorders and neurodegenerative diseases, may be associated with failure to process genomic Ribonucleotides Genomic Ribonucleotides function as a strand-discrimination signal during DNA mismatch repair, and they may have other physiological roles. Ribonucleotides are incorporated into DNA by various mechanisms, including by DNA polymerases during replication. Such Ribonucleotides may have physiological functions, but their presence is typically associated with diverse structural aberrations and interferes with fundamental processes, including DNA replication, repair and transcription. Thus, efficient mechanisms of ribonucleotide removal are key to maintaining genomic integrity and functionality.
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Processing Ribonucleotides incorporated during eukaryotic DNA replication.
Nature reviews. Molecular cell biology, 2016Co-Authors: Jessica S Williams, Scott A Lujan, Thomas A. KunkelAbstract:The information encoded in DNA is influenced by the presence of non-canonical nucleotides, the most frequent of which are Ribonucleotides. In this Review, we discuss recent discoveries about ribonucleotide incorporation into DNA during replication by the three major eukaryotic replicases, DNA polymerases α, δ and e. The presence of Ribonucleotides in DNA causes short deletion mutations and may result in the generation of single- and double-strand DNA breaks, leading to genome instability. We describe how these Ribonucleotides are removed from DNA through ribonucleotide excision repair and by topoisomerase I. We discuss the biological consequences and the physiological roles of Ribonucleotides in DNA, and consider how deficiencies in their removal from DNA may be important in the aetiology of disease.
Andrei Chabes - One of the best experts on this subject based on the ideXlab platform.
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Ribonucleotides incorporated by the yeast mitochondrial DNA polymerase are not repaired.
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Paulina H. Wanrooij, Anders R Clausen, Martin K. M. Engqvist, Josefin M. E. Forslund, Clara Navarrete, Anna Karin Nilsson, Juhan Sedman, Sjoerd Wanrooij, Andrei ChabesAbstract:Incorporation of Ribonucleotides into DNA during genome replication is a significant source of genomic instability. The frequency of Ribonucleotides in DNA is determined by deoxyribonucleoside triphosphate/ribonucleoside triphosphate (dNTP/rNTP) ratios, by the ability of DNA polymerases to discriminate against Ribonucleotides, and by the capacity of repair mechanisms to remove incorporated Ribonucleotides. To simultaneously compare how the nuclear and mitochondrial genomes incorporate and remove Ribonucleotides, we challenged these processes by changing the balance of cellular dNTPs. 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 removal of Ribonucleotides incorporated by the mtDNA polymerase. Furthermore, as cytosolic dNTP pool imbalances were transmitted equally well into the nucleus and the mitochondria, our results support a view of the cytosolic and mitochondrial dNTP pools in frequent exchange.
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Evidence that processing of Ribonucleotides in DNA by topoisomerase 1 is leading-strand specific
Nature Structural & Molecular Biology, 2015Co-Authors: Jessica S Williams, Anders R Clausen, Scott A Lujan, Alan B Clark, Andrei Chabes, Lisette Marjavaara, Peter M Burgers, Thomas A. KunkelAbstract:Ribonucleotides incorporated during DNA replication are removed by RNase H2–dependent ribonucleotide excision repair (RER). In RER-defective yeast, topoisomerase 1 (Top1) incises DNA at unrepaired Ribonucleotides, initiating their removal, but this is accompanied by RNA-DNA–damage phenotypes. Here we show that these phenotypes are incurred by a high level of Ribonucleotides incorporated by a leading strand–replicase variant, DNA polymerase (Pol) ɛ, but not by orthologous variants of the lagging-strand replicases, Pols α or δ. Moreover, loss of both RNases H1 and H2 is lethal in combination with increased ribonucleotide incorporation by Pol ɛ but not by Pols α or δ. Several explanations for this asymmetry are considered, including the idea that Top1 incision at Ribonucleotides relieves torsional stress in the nascent leading strand but not in the nascent lagging strand, in which preexisting nicks prevent the accumulation of superhelical tension. In the absence of RNase H2, Ribonucleotides incorporated during DNA replication can be processed by Top1. This activity is directed to the nascent leading strand, because gaps in the lagging strand would limit torsional tension.
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evidence that processing of Ribonucleotides in dna by topoisomerase 1 is leading strand specific
Nature Structural & Molecular Biology, 2015Co-Authors: Jessica S Williams, Anders R Clausen, Scott A Lujan, Alan B Clark, Andrei Chabes, Lisette Marjavaara, Peter M. J. Burgers, Thomas A. KunkelAbstract:Ribonucleotides incorporated during DNA replication are removed by RNase H2-dependent ribonucleotide excision repair (RER). In RER-defective yeast, topoisomerase 1 (Top1) incises DNA at unrepaired ...
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Topoisomerase 1-mediated removal of Ribonucleotides from nascent leading-strand DNA.
Molecular cell, 2013Co-Authors: Jessica S Williams, Scott A Lujan, Andrei Chabes, Dana J. Smith, Lisette Marjavaara, Thomas A. KunkelAbstract:RNase H2-dependent ribonucleotide excision repair (RER) removes Ribonucleotides incorporated during DNA replication. When RER is defective, Ribonucleotides in the nascent leading strand of the yeas ...
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Genome instability due to ribonucleotide incorporation into DNA
Nature Chemical Biology, 2010Co-Authors: Stephanie A Nick Mcelhinny, Dinesh Kumar, Alan B Clark, Danielle L Watt, Brian E Watts, Else-britt Lundström, Erik Johansson, Andrei Chabes, Thomas A. KunkelAbstract:Polymerase exclusion of Ribonucleotides during DNA replication is imperfect. New data indicate that DNA polymerase ϵ incorporates into DNA Ribonucleotides that are repaired by an RNase H2–dependent process and that defective repair results in replicative stress and genome instability. Maintaining the chemical identity of DNA depends on ribonucleotide exclusion by DNA polymerases. However, ribonucleotide exclusion during DNA synthesis in vitro is imperfect. To determine whether Ribonucleotides are incorporated during DNA replication in vivo , we substituted leucine or glycine for an active-site methionine in yeast DNA polymerase ϵ (Pol ϵ). Ribonucleotide incorporation in vitro was three-fold lower for M644L and 11-fold higher for M644G Pol ϵ compared to wild-type Pol ϵ. This hierarchy was recapitulated in vivo in yeast strains lacking RNase H2. Moreover, the pol2-M644G rnh201 Δ strain progressed more slowly through S phase, had elevated dNTP pools and generated 2–5-base-pair deletions in repetitive sequences at a high rate and in a gene orientation–dependent manner. The data indicate that Ribonucleotides are incorporated during replication in vivo , that they are removed by RNase H2–dependent repair and that defective repair results in replicative stress and genome instability via DNA strand misalignment.
