29 results on '"Daigaku Y"'
Search Results
2. Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle
- Author
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Daigaku, Y., Mashiko, S., Mishiba, K., Yamamura, S., Ui, A., Enomoto, T., and Yamamoto, K.
- Abstract
A CAN1/can1@D heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.
- Published
- 2006
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3. Bulk synthesis and beyond: The roles of eukaryotic replicative DNA polymerases.
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Bainbridge LJ and Daigaku Y
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- Humans, DNA metabolism, DNA biosynthesis, DNA-Directed DNA Polymerase metabolism, Animals, DNA Polymerase II metabolism, Eukaryota enzymology, Eukaryota genetics, DNA Polymerase III metabolism, Eukaryotic Cells metabolism, Eukaryotic Cells enzymology, DNA Polymerase I metabolism, DNA Replication
- Abstract
An organism's genomic DNA must be accurately duplicated during each cell cycle. DNA synthesis is catalysed by DNA polymerase enzymes, which extend nucleotide polymers in a 5' to 3' direction. This inherent directionality necessitates that one strand is synthesised forwards (leading), while the other is synthesised backwards discontinuously (lagging) to couple synthesis to the unwinding of duplex DNA. Eukaryotic cells possess many diverse polymerases that coordinate to replicate DNA, with the three main replicative polymerases being Pol α, Pol δ and Pol ε. Studies conducted in yeasts and human cells utilising mutant polymerases that incorporate molecular signatures into nascent DNA implicate Pol ε in leading strand synthesis and Pol α and Pol δ in lagging strand replication. Recent structural insights have revealed how the spatial organization of these enzymes around the core helicase facilitates their strand-specific roles. However, various challenging situations during replication require flexibility in the usage of these enzymes, such as during replication initiation or encounters with replication-blocking adducts. This review summarises the roles of the replicative polymerases in bulk DNA replication and explores their flexible and dynamic deployment to complete genome replication. We also examine how polymerase usage patterns can inform our understanding of global replication dynamics by revealing replication fork directionality to identify regions of replication initiation and termination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
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4. Strategic targeting of Cas9 nickase induces large segmental duplications.
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Sugiyama Y, Okada S, Daigaku Y, Kusumoto E, and Ito T
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- Humans, DNA Replication, CRISPR-Associated Protein 9 genetics, CRISPR-Associated Protein 9 metabolism, Gene Duplication, Replication Origin genetics, DNA Breaks, Double-Stranded, CRISPR-Cas Systems genetics, Saccharomyces cerevisiae genetics
- Abstract
Gene/segmental duplications play crucial roles in genome evolution and variation. Here, we introduce paired nicking-induced amplification (PNAmp) for their experimental induction. PNAmp strategically places two Cas9 nickases upstream and downstream of a replication origin on opposite strands. This configuration directs the sister replication forks initiated from the origin to break at the nicks, generating a pair of one-ended double-strand breaks. If homologous sequences flank the two break sites, then end resection converts them to single-stranded DNAs that readily anneal to drive duplication of the region bounded by the homologous sequences. PNAmp induces duplication of segments as large as ∼1 Mb with efficiencies exceeding 10% in the budding yeast Saccharomyces cerevisiae. Furthermore, appropriate splint DNAs allow PNAmp to duplicate/multiplicate even segments not bounded by homologous sequences. We also provide evidence for PNAmp in mammalian cells. Therefore, PNAmp provides a prototype method to induce structural variations by manipulating replication fork progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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5. Adaptive use of error-prone DNA polymerases provides flexibility in genome replication during tumorigenesis.
- Author
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Bainbridge LJ and Daigaku Y
- Subjects
- Humans, Animals, Proliferating Cell Nuclear Antigen metabolism, Proliferating Cell Nuclear Antigen genetics, Ubiquitination, Mutagenesis, DNA Repair genetics, DNA Replication, DNA-Directed DNA Polymerase metabolism, DNA-Directed DNA Polymerase genetics, Carcinogenesis genetics, Neoplasms genetics, Neoplasms pathology
- Abstract
Human cells possess many different polymerase enzymes, which collaborate in conducting DNA replication and genome maintenance to ensure faithful duplication of genetic material. Each polymerase performs a specialized role, together providing a balance of accuracy and flexibility to the replication process. Perturbed replication increases the requirement for flexibility to ensure duplication of the entire genome. Flexibility is provided via the use of error-prone polymerases, which maintain the progression of challenged DNA replication at the expense of mutagenesis, an enabling characteristic of cancer. This review describes our recent understanding of mechanisms that alter the usage of polymerases during tumorigenesis and examines the implications of this for cell survival and tumor progression. Although expression levels of polymerases are often misregulated in cancers, this does not necessarily alter polymerase usage since an additional regulatory step may govern the use of these enzymes. We therefore also examine how the regulatory mechanisms of DNA polymerases, such as Rad18-mediated PCNA ubiquitylation, may impact the functionalization of error-prone polymerases to tolerate oncogene-induced replication stress. Crucially, it is becoming increasingly evident that cancer cells utilize error-prone polymerases to sustain ongoing replication in response to oncogenic mutations which inactivate key DNA replication and repair pathways, such as BRCA deficiency. This accelerates mutagenesis and confers chemoresistance, but also presents a dependency that can potentially be exploited by therapeutics., (© 2024 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)
- Published
- 2024
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6. NELF coordinates Pol II transcription termination and DNA replication initiation.
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Nakayama C, Daigaku Y, Aoi Y, Fang Q, Kimura H, Shilatifard A, Tellier M, and Nojima T
- Abstract
Regulation of RNA polymerase II (Pol II) transcription is closely associated with cell proliferation. However, it remains unclear how the Pol II transcription program is altered in cancer to favour cell growth. Here, we find that gene expression of NELFCD , a known negative elongation factor, is up-regulated in colorectal tumours. To dissect the direct role of NELF-C on Pol II transcription in such cancer, we employed an auxin-dependent protein degradation system for NELF-C in combination with nascent transcript sequencing technologies. Strikingly, we demonstrated that the acute loss of NELF-C protein globally perturbs Pol II transcription termination and also increases transcription elongation rate, independently of promoter-proximal Pol II pausing. This results in Pol II transcription into DNA replication initiation zones, and may link to failure of the cell cycle transition into S phase. We anticipate that NELF will be a potential therapeutic target to restrict colorectal cancers by promoting transcription-replication conflict., Highlights: Expression of NELFCD transcript is up-regulated in colorectal tumors NELF-C protein is mandatory for the transition between G1-S phases during cell cycleNELF-C loss impairs transcription termination independently of Pol II promoter-proximal pausingNELF-C loss leads Pol II to invade DNA replication initiation zones.
- Published
- 2024
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7. Onset model of mutually catalytic self-replicative systems formed by an assembly of polynucleotides.
- Author
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Sawada Y, Daigaku Y, and Toma K
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- DNA genetics, Polynucleotides genetics, RNA genetics
- Abstract
Self-replicability is a unique attribute observed in all living organisms, and the question of how the life was physically initiated could be equivalent to the question of how self-replicating informative polymers were formed in the abiotic material world. It has been suggested that the present DNA and proteins world was preceded by an RNA world in which genetic information of RNA molecules was replicated by the mutual catalytic function of RNA molecules. However, the important question of how the transition occurred from a material world to the very early pre-RNA world remains unsolved both experimentally and theoretically. We present an onset model of mutually catalytic self-replicative systems formed in an assembly of polynucleotides. A quantitative expression of the critical condition for the onset of growing fluctuation towards self-replication in this model is obtained by analytical and numerical calculations.
- Published
- 2023
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8. Novel compound heterozygous mutations in UHRF1 are associated with atypical immunodeficiency, centromeric instability and facial anomalies syndrome with distinctive genome-wide DNA hypomethylation.
- Author
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Unoki M, Velasco G, Kori S, Arita K, Daigaku Y, Yeung WKA, Fujimoto A, Ohashi H, Kubota T, Miyake K, and Sasaki H
- Subjects
- Humans, CCAAT-Enhancer-Binding Proteins genetics, CCAAT-Enhancer-Binding Proteins metabolism, DNA genetics, DNA metabolism, DNA Methylation genetics, DNA Methylation physiology, HEK293 Cells, Immunologic Deficiency Syndromes genetics, Immunologic Deficiency Syndromes metabolism, Mutation, Ubiquitin-Protein Ligases genetics, Ubiquitin-Protein Ligases metabolism, Chromosomal Instability genetics, Chromosomal Instability physiology, Centromere genetics, Centromere metabolism, Face abnormalities, Genome, Human genetics, Genome, Human physiology, Histones genetics, Histones metabolism, Primary Immunodeficiency Diseases genetics, Primary Immunodeficiency Diseases metabolism
- Abstract
Immunodeficiency, centromeric instability and facial anomalies (ICF) syndrome is in most cases caused by mutations in either DNA methyltransferase (DNMT)3B, zinc finger and BTB domain containing 24, cell division cycle associated 7 or helicase lymphoid-specific. However, the causative genes of a few ICF patients remain unknown. We, herein, identified ubiquitin-like with plant homeodomain and really interesting new gene finger domains 1 (UHRF1) as a novel causative gene of one such patient with atypical symptoms. This patient is a compound heterozygote for two previously unreported mutations in UHRF1: c.886C > T (p.R296W) and c.1852C > T (p.R618X). The R618X mutation plausibly caused nonsense-mediated decay, while the R296W mutation changed the higher order structure of UHRF1, which is indispensable for the maintenance of CG methylation along with DNMT1. Genome-wide methylation analysis revealed that the patient had a centromeric/pericentromeric hypomethylation, which is the main ICF signature, but also had a distinctive hypomethylation pattern compared to patients with the other ICF syndrome subtypes. Structural and biochemical analyses revealed that the R296W mutation disrupted the protein conformation and strengthened the binding affinity of UHRF1 with its partner LIG1 and reduced ubiquitylation activity of UHRF1 towards its ubiquitylation substrates, histone H3 and proliferating cell nuclear antigen -associated factor 15 (PAF15). We confirmed that the R296W mutation causes hypomethylation at pericentromeric repeats by generating the HEK293 cell lines that mimic the patient's UHRF1 molecular context. Since proper interactions of the UHRF1 with LIG1, PAF15 and histone H3 are essential for the maintenance of CG methylation, the mutation could disturb the maintenance process. Evidence for the importance of the UHRF1 conformation for CG methylation in humans is, herein, provided for the first time and deepens our understanding of its role in regulation of CG methylation., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)
- Published
- 2023
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9. Cryo-EM structures of human SPCA1a reveal the mechanism of Ca 2+ /Mn 2+ transport into the Golgi apparatus.
- Author
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Chen Z, Watanabe S, Hashida H, Inoue M, Daigaku Y, Kikkawa M, and Inaba K
- Subjects
- Humans, Cryoelectron Microscopy, Binding Sites, Adenosine Triphosphate, Calcium-Transporting ATPases, Golgi Apparatus, Adenosine Triphosphatases
- Abstract
Secretory pathway Ca
2+ /Mn2+ ATPase 1 (SPCA1) actively transports cytosolic Ca2+ and Mn2+ into the Golgi lumen, playing a crucial role in cellular calcium and manganese homeostasis. Detrimental mutations of the ATP2C1 gene encoding SPCA1 cause Hailey-Hailey disease. Here, using nanobody/megabody technologies, we determined cryo-electron microscopy structures of human SPCA1a in the ATP and Ca2+ /Mn2+ -bound (E1-ATP) state and the metal-free phosphorylated (E2P) state at 3.1- to 3.3-Å resolutions. The structures revealed that Ca2+ and Mn2+ share the same metal ion-binding pocket with similar but notably different coordination geometries in the transmembrane domain, corresponding to the second Ca2+ -binding site in sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA). In the E1-ATP to E2P transition, SPCA1a undergoes similar domain rearrangements to those of SERCA. Meanwhile, SPCA1a shows larger conformational and positional flexibility of the second and sixth transmembrane helices, possibly explaining its wider metal ion specificity. These structural findings illuminate the unique mechanisms of SPCA1a-mediated Ca2+ /Mn2+ transport.- Published
- 2023
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10. Global landscape of replicative DNA polymerase usage in the human genome.
- Author
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Koyanagi E, Kakimoto Y, Minamisawa T, Yoshifuji F, Natsume T, Higashitani A, Ogi T, Carr AM, Kanemaki MT, and Daigaku Y
- Subjects
- Humans, DNA Replication genetics, Genome, Human genetics, DNA-Directed DNA Polymerase genetics, DNA-Directed DNA Polymerase metabolism
- Abstract
The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequencing (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combining the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality plus zones of replication initiation and termination. We confirm that transcriptional activity contributes to the pattern of initiation and termination and, by separately analysing the effect of transcription on co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands is compromised by transcription in both co-directional and convergent forks. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability. Together, our result demonstrated that Pu-seq in human cells provides a powerful and straightforward methodology to explore DNA polymerase usage and replication fork dynamics., (© 2022. The Author(s).)
- Published
- 2022
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11. Ubiquitination of DNA Damage-Stalled RNAPII Promotes Transcription-Coupled Repair.
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Nakazawa Y, Hara Y, Oka Y, Komine O, van den Heuvel D, Guo C, Daigaku Y, Isono M, He Y, Shimada M, Kato K, Jia N, Hashimoto S, Kotani Y, Miyoshi Y, Tanaka M, Sobue A, Mitsutake N, Suganami T, Masuda A, Ohno K, Nakada S, Mashimo T, Yamanaka K, Luijsterburg MS, and Ogi T
- Subjects
- Animals, Carrier Proteins genetics, Carrier Proteins metabolism, DNA metabolism, DNA Damage physiology, DNA Helicases metabolism, DNA Repair Enzymes genetics, DNA Repair Enzymes metabolism, Female, HCT116 Cells, HEK293 Cells, HeLa Cells, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, RNA Polymerase II genetics, Ubiquitination, DNA Repair physiology, RNA Polymerase II metabolism
- Abstract
Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)
- Published
- 2020
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12. Vital roles of PCNA K165 modification during C. elegans gametogenesis and embryogenesis.
- Author
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Shao Z, Niwa S, Higashitani A, and Daigaku Y
- Subjects
- Animals, Caenorhabditis elegans physiology, Caenorhabditis elegans Proteins genetics, DNA Damage, Epistasis, Genetic, Ubiquitin-Conjugating Enzymes genetics, Amino Acid Substitution, Caenorhabditis elegans embryology, Caenorhabditis elegans genetics, Embryonic Development genetics, Gametogenesis genetics, Proliferating Cell Nuclear Antigen genetics
- Abstract
In eukaryotes, the DNA damage bypass pathway is promoted by ubiquitylation of PCNA at the conserved lysine 164. Using CRISPR-Cas9 system, we introduced amino acid substitution at K165 of C. elegans PCNA that corresponds to K164 in other characterised organisms and examined the contribution of this residue at a variety of stages during development. In the presence of UV-induced DNA lesions, PCNA-K165 is crucial for not only the early embryonic stages but also during larval development, implicating its functions for a broad time period during animal development. We also show that, without induction of DNA damage, concomitant inhibition of PCNA ubiquitylation and checkpoint activation causes abnormal gametogenesis events and severely impairs reproduction of worms. Our findings suggest a conserved function of PCNA ubiquitylation in tolerance of UV-induced damage and also propose that PCNA ubiquitylation contributes to gametogenesis during unperturbed C. elegans development., (Copyright © 2019 Elsevier B.V. All rights reserved.)
- Published
- 2019
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13. Spatial separation between replisome- and template-induced replication stress signaling.
- Author
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García-Rodríguez N, Morawska M, Wong RP, Daigaku Y, and Ulrich HD
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- Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, DNA, Fungal genetics, Exodeoxyribonucleases genetics, Exodeoxyribonucleases metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Cell Cycle Checkpoints physiology, DNA Replication physiology, DNA, Fungal biosynthesis, S Phase physiology, Saccharomyces cerevisiae metabolism
- Abstract
Polymerase-blocking DNA lesions are thought to elicit a checkpoint response via accumulation of single-stranded DNA at stalled replication forks. However, as an alternative to persistent fork stalling, re-priming downstream of lesions can give rise to daughter-strand gaps behind replication forks. We show here that the processing of such structures by an exonuclease, Exo1, is required for timely checkpoint activation, which in turn prevents further gap erosion in S phase. This Rad9-dependent mechanism of damage signaling is distinct from the Mrc1-dependent, fork-associated response to replication stress induced by conditions such as nucleotide depletion or replisome-inherent problems, but reminiscent of replication-independent checkpoint activation by single-stranded DNA Our results indicate that while replisome stalling triggers a checkpoint response directly at the stalled replication fork, the response to replication stress elicited by polymerase-blocking lesions mainly emanates from Exo1-processed, postreplicative daughter-strand gaps, thus offering a mechanistic explanation for the dichotomy between replisome- versus template-induced checkpoint signaling., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)
- Published
- 2018
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14. Analysis of Replicative Polymerase Usage by Ribonucleotide Incorporation.
- Author
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Keszthelyi A, Miyabe I, Ptasińska K, Daigaku Y, Naiman K, and Carr AM
- Subjects
- Computational Biology methods, DNA Cleavage, DNA, Fungal, Genome, Fungal, High-Throughput Nucleotide Sequencing, Replication Origin, Saccharomyces cerevisiae genetics, Software, DNA Replication, DNA-Directed DNA Polymerase metabolism, Ribonucleotides
- Abstract
Mapping the usage of replicative DNA polymerases has previously proved to be technically challenging. By exploiting mutant polymerases that incorporate ribonucleotides into the DNA with a significantly higher proficiency than their wild-type counterparts, we and others have developed methods that can identify what proportion of each DNA strand (i.e., the Watson and Crick strands) is replicated by a specific DNA polymerase. The incorporation of excess ribonucleotides by a mutated polymerase effectively marks, in each individual cells, the DNA strand that is replicated by that specific mutated polymerase. Changes to DNA polymerase usage can be examined at specific loci by Southern blot analysis while a global analysis of polymerase usage can be achieved by applying next-generation sequencing. This genome-wide data also provides a direct measure of replication origin efficiency and can be used to indirectly calculate replication timing.
- Published
- 2018
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15. PCNA ubiquitylation ensures timely completion of unperturbed DNA replication in fission yeast.
- Author
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Daigaku Y, Etheridge TJ, Nakazawa Y, Nakayama M, Watson AT, Miyabe I, Ogi T, Osborne MA, and Carr AM
- Subjects
- Cell Line, Tumor, Chromatin genetics, Chromatin metabolism, DNA-Directed DNA Polymerase genetics, DNA-Directed DNA Polymerase metabolism, Humans, Proliferating Cell Nuclear Antigen genetics, Schizosaccharomyces metabolism, DNA Replication, Proliferating Cell Nuclear Antigen metabolism, Schizosaccharomyces genetics, Ubiquitination
- Abstract
PCNA ubiquitylation on lysine 164 is required for DNA damage tolerance. In many organisms PCNA is also ubiquitylated in unchallenged S phase but the significance of this has not been established. Using Schizosaccharomyces pombe, we demonstrate that lysine 164 ubiquitylation of PCNA contributes to efficient DNA replication in the absence of DNA damage. Loss of PCNA ubiquitylation manifests most strongly at late replicating regions and increases the frequency of replication gaps. We show that PCNA ubiquitylation increases the proportion of chromatin associated PCNA and the co-immunoprecipitation of Polymerase δ with PCNA during unperturbed replication and propose that ubiquitylation acts to prolong the chromatin association of these replication proteins to allow the efficient completion of Okazaki fragment synthesis by mediating gap filling.
- Published
- 2017
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16. Polymerase δ replicates both strands after homologous recombination-dependent fork restart.
- Author
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Miyabe I, Mizuno K, Keszthelyi A, Daigaku Y, Skouteri M, Mohebi S, Kunkel TA, Murray JM, and Carr AM
- Subjects
- Cell Division, Schizosaccharomyces genetics, Schizosaccharomyces physiology, DNA Polymerase III metabolism, DNA Replication, Homologous Recombination, Schizosaccharomyces enzymology
- Abstract
To maintain genetic stability, DNA must be replicated only once per cell cycle, and replication must be completed even when individual replication forks are inactivated. Because fork inactivation is common, passive convergence of an adjacent fork is insufficient to rescue all inactive forks. Thus, eukaryotic cells have evolved homologous recombination-dependent mechanisms to restart persistent inactive forks. Completing DNA synthesis via homologous recombination-restarted replication (HoRReR) ensures cell survival, but at a cost. One such cost is increased mutagenesis because HoRReR is more error prone than canonical replication. This increased error rate implies the HoRReR mechanism is distinct from that of a canonical fork. Here we demonstrate, in Schizosaccharomyces pombe, that a DNA sequence duplicated by HoRReR during S phase is replicated semiconservatively, but both the leading and lagging strands are synthesized by DNA polymerase δ.
- Published
- 2015
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17. Mapping ribonucleotides in genomic DNA and exploring replication dynamics by polymerase usage sequencing (Pu-seq).
- Author
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Keszthelyi A, Daigaku Y, Ptasińska K, Miyabe I, and Carr AM
- Subjects
- DNA Replication, DNA chemistry, DNA-Directed DNA Polymerase metabolism, Genomics methods, High-Throughput Nucleotide Sequencing methods, Ribonucleotides analysis
- Abstract
Ribonucleotides are frequently misincorporated into DNA during replication, and they are rapidly repaired by ribonucleotide excision repair (RER). Although ribonucleotides in template DNA perturb replicative polymerases and can be considered as DNA damage, they also serve positive biological functions, including directing the orientation of mismatch repair. Here we describe a method for ribonucleotide identification by high-throughput sequencing that allows mapping of the location of ribonucleotides across the genome. When combined with specific mutations in the replicative polymerases that incorporate ribonucleotides at elevated frequencies, our ribonucleotide identification method was adapted to map polymerase usage across the genome. Polymerase usage sequencing (Pu-seq) has been used to define, in unprecedented detail, replication dynamics in yeasts. Although other methods that examine replication dynamics provide direct measures of replication timing and indirect estimates of origin efficiency, Pu-seq directly ascertains origin efficiency. The Pu-seq protocol can be completed in 12-14 d.
- Published
- 2015
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18. A global profile of replicative polymerase usage.
- Author
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Daigaku Y, Keszthelyi A, Müller CA, Miyabe I, Brooks T, Retkute R, Hubank M, Nieduszynski CA, and Carr AM
- Subjects
- DNA chemistry, Replication Origin, DNA Polymerase I physiology, DNA Polymerase II physiology, DNA Polymerase III physiology, DNA Replication physiology, Models, Genetic, Schizosaccharomyces genetics
- Abstract
Three eukaryotic DNA polymerases are essential for genome replication. Polymerase (Pol) α-primase initiates each synthesis event and is rapidly replaced by processive DNA polymerases: Polɛ replicates the leading strand, whereas Polδ performs lagging-strand synthesis. However, it is not known whether this division of labor is maintained across the whole genome or how uniform it is within single replicons. Using Schizosaccharomyces pombe, we have developed a polymerase usage sequencing (Pu-seq) strategy to map polymerase usage genome wide. Pu-seq provides direct replication-origin location and efficiency data and indirect estimates of replication timing. We confirm that the division of labor is broadly maintained across an entire genome. However, our data suggest a subtle variability in the usage of the two polymerases within individual replicons. We propose that this results from occasional leading-strand initiation by Polδ followed by exchange for Polɛ.
- Published
- 2015
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19. Quantification of DNA-associated proteins inside eukaryotic cells using single-molecule localization microscopy.
- Author
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Etheridge TJ, Boulineau RL, Herbert A, Watson AT, Daigaku Y, Tucker J, George S, Jönsson P, Palayret M, Lando D, Laue E, Osborne MA, Klenerman D, Lee SF, and Carr AM
- Subjects
- Cell Cycle, DNA Replication, Diffusion, Minichromosome Maintenance Complex Component 4 analysis, Proliferating Cell Nuclear Antigen analysis, Schizosaccharomyces genetics, Schizosaccharomyces pombe Proteins analysis, DNA-Binding Proteins analysis, Microscopy, Fluorescence methods
- Abstract
Development of single-molecule localization microscopy techniques has allowed nanometre scale localization accuracy inside cells, permitting the resolution of ultra-fine cell structure and the elucidation of crucial molecular mechanisms. Application of these methodologies to understanding processes underlying DNA replication and repair has been limited to defined in vitro biochemical analysis and prokaryotic cells. In order to expand these techniques to eukaryotic systems, we have further developed a photo-activated localization microscopy-based method to directly visualize DNA-associated proteins in unfixed eukaryotic cells. We demonstrate that motion blurring of fluorescence due to protein diffusivity can be used to selectively image the DNA-bound population of proteins. We designed and tested a simple methodology and show that it can be used to detect changes in DNA binding of a replicative helicase subunit, Mcm4, and the replication sliding clamp, PCNA, between different stages of the cell cycle and between distinct genetic backgrounds., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2014
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20. Optimisation of the Schizosaccharomyces pombe urg1 expression system.
- Author
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Watson AT, Daigaku Y, Mohebi S, Etheridge TJ, Chahwan C, Murray JM, and Carr AM
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- Arginine pharmacology, Endonucleases metabolism, Gene Expression drug effects, Genetic Loci genetics, Hydroxyurea pharmacology, Indoleacetic Acids pharmacology, Phenotype, Plasmids genetics, Promoter Regions, Genetic genetics, RNA Stability, RNA, Messenger chemistry, RNA, Messenger genetics, RNA, Messenger metabolism, Schizosaccharomyces drug effects, Transcription, Genetic drug effects, mRNA Cleavage and Polyadenylation Factors genetics, Genetic Engineering methods, Schizosaccharomyces genetics, Schizosaccharomyces pombe Proteins genetics
- Abstract
The ability to study protein function in vivo often relies on systems that regulate the presence and absence of the protein of interest. Two limitations for previously described transcriptional control systems that are used to regulate protein expression in fission yeast are: the time taken for inducing conditions to initiate transcription and the ability to achieve very low basal transcription in the "OFF-state". In previous work, we described a Cre recombination-mediated system that allows the rapid and efficient regulation of any gene of interest by the urg1 promoter, which has a dynamic range of approximately 75-fold and which is induced within 30-60 minutes of uracil addition. In this report we describe easy-to-use and versatile modules that can be exploited to significantly tune down Purg1 "OFF-levels" while maintaining an equivalent dynamic range. We also provide plasmids and tools for combining Purg1 transcriptional control with the auxin degron tag to help maintain a null-like phenotype. We demonstrate the utility of this system by improved regulation of HO-dependent site-specific DSB formation, by the regulation Rtf1-dependent replication fork arrest and by controlling Rhp18(Rad18)-dependent post replication repair.
- Published
- 2013
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21. Action spectrum analysis of UVR genotoxicity for skin: the border wavelengths between UVA and UVB can bring serious mutation loads to skin.
- Author
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Ikehata H, Higashi S, Nakamura S, Daigaku Y, Furusawa Y, Kamei Y, Watanabe M, Yamamoto K, Hieda K, Munakata N, and Ono T
- Subjects
- Animals, Apoptosis radiation effects, DNA Damage genetics, DNA Damage radiation effects, Disease Models, Animal, Dose-Response Relationship, Radiation, Erythema epidemiology, Lac Operon genetics, Mice, Mice, Transgenic, Risk Factors, Skin pathology, Erythema etiology, Erythema genetics, Mutation genetics, Skin radiation effects, Spectrum Analysis, Ultraviolet Rays adverse effects
- Abstract
UVR causes erythema, which has been used as a standardized index to evaluate the risk of UVR for human skin. However, the genotoxic significance of erythema has not been elucidated clearly. Here, we characterized the wavelength dependence of the genotoxic and erythematic effects of UVR for the skin by analyzing the induction kinetics of mutation and inflammation in mouse skin using lacZ-transgenic mice and monochromatic UVR sources. We determined their action spectra and found a close correlation between erythema and an epidermis-specific antigenotoxic response, mutation induction suppression (MIS), which suppressed the mutant frequencies (MFs) to a constant plateau level only 2-3-fold higher than the background MF at the cost of apoptotic cell death, suggesting that erythema may represent the threshold beyond which the antigenotoxic but tissue-destructive MIS response commences. However, we unexpectedly found that MIS attenuates remarkably at the border wavelengths between UVA and UVB around 315 nm, elevating the MF plateaus up to levels ∼40-fold higher than the background level. Thus, these border wavelengths can bring heavier mutation loads to the skin than the otherwise more mutagenic and erythematic shorter wavelengths, suggesting that erythema-based UVR risk evaluation should be reconsidered.
- Published
- 2013
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22. Ubiquitin-dependent DNA damage bypass is separable from genome replication.
- Author
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Daigaku Y, Davies AA, and Ulrich HD
- Subjects
- Cell Cycle physiology, Chromatin metabolism, DNA Damage radiation effects, Proliferating Cell Nuclear Antigen metabolism, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins, Time Factors, Ubiquitin-Conjugating Enzymes, Ultraviolet Rays, DNA Damage genetics, DNA Replication genetics, Genome genetics, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae genetics, Ubiquitin metabolism
- Abstract
Post-replication repair (PRR) is a pathway that allows cells to bypass or overcome lesions during DNA replication. In eukaryotes, damage bypass is activated by ubiquitylation of the replication clamp PCNA through components of the RAD6 pathway. Whereas monoubiquitylation of PCNA allows mutagenic translesion synthesis by damage-tolerant DNA polymerases, polyubiquitylation is required for an error-free pathway that probably involves a template switch to the undamaged sister chromatid. Both the timing of PRR events during the cell cycle and their location relative to replication forks, as well as the factors required downstream of PCNA ubiquitylation, have remained poorly characterized. Here we demonstrate that the RAD6 pathway normally operates during S phase. However, using an inducible system of DNA damage bypass in budding yeast (Saccharomyces cerevisiae), we show that the process is separable in time and space from genome replication, thus allowing direct visualization and quantification of productive PRR tracts. We found that both during and after S phase ultraviolet-radiation-induced lesions are bypassed predominantly via translesion synthesis, whereas the error-free pathway functions as a backup system. Our approach has revealed the distribution of PRR tracts in a synchronized cell population. It will allow an in-depth mechanistic analysis of how cells manage the processing of lesions to their genomes during and after replication.
- Published
- 2010
- Full Text
- View/download PDF
23. Activation of ubiquitin-dependent DNA damage bypass is mediated by replication protein a.
- Author
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Davies AA, Huttner D, Daigaku Y, Chen S, and Ulrich HD
- Subjects
- Animals, Cell Cycle physiology, Cell Line, DNA Replication, DNA, Single-Stranded genetics, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, Humans, Proliferating Cell Nuclear Antigen genetics, Proliferating Cell Nuclear Antigen metabolism, Replication Protein A genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Two-Hybrid System Techniques, Ubiquitin genetics, DNA Damage, DNA, Single-Stranded metabolism, Replication Protein A metabolism, Ubiquitin metabolism
- Abstract
Replicative DNA damage bypass, mediated by the ubiquitylation of the sliding clamp protein PCNA, facilitates the survival of a cell in the presence of genotoxic agents, but it can also promote genomic instability by damage-induced mutagenesis. We show here that PCNA ubiquitylation in budding yeast is activated independently of the replication-dependent S phase checkpoint but by similar conditions involving the accumulation of single-stranded DNA at stalled replication intermediates. The ssDNA-binding replication protein A (RPA), an essential complex involved in most DNA transactions, is required for damage-induced PCNA ubiquitylation. We found that RPA directly interacts with the ubiquitin ligase responsible for the modification of PCNA, Rad18, both in yeast and in mammalian cells. Association of the ligase with chromatin is detected where RPA is most abundant, and purified RPA can recruit Rad18 to ssDNA in vitro. Our results therefore implicate the RPA complex in the activation of DNA damage tolerance.
- Published
- 2008
- Full Text
- View/download PDF
24. Cytosolic LC3 ratio as a sensitive index of macroautophagy in isolated rat hepatocytes and H4-II-E cells.
- Author
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Karim MR, Kanazawa T, Daigaku Y, Fujimura S, Miotto G, and Kadowaki M
- Subjects
- Animals, Biomarkers metabolism, Blotting, Western, Carcinoma, Hepatocellular pathology, Cell Line, Tumor, Hydrolysis, Liver Neoplasms pathology, Male, Models, Biological, Rats, Rats, Wistar, Sensitivity and Specificity, Subcellular Fractions metabolism, Autophagy, Cytosol metabolism, Hepatocytes metabolism, Microtubule-Associated Proteins metabolism
- Abstract
Macroautophagy, an intracellular bulk degradation process in eukaryotes, is sensitive to nutrient supply and deprivation. Microtubule-associated protein 1 light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an indispensable role in macroautophagy formation and is a suitable marker for this process. Through analysis of the subcellular distribution of LC3, we determined that the cytosolic fraction contained not only a precursor form (LC3-I), but also an apparent active form (LC3-IIs). Both cytosolic LC3-I and LC3-IIs were more responsive to amino acids than those of total homogenate. Moreover, changes in the LC3-IIs/I ratio reflected those in the total proteolytic flux remarkably in both fresh rat hepatocytes and H4-II-E cell lines. Thus, in addition to a sensitive index of macroautophagy, calculating the cytosolic LC3 ratio became an easy and quick quantitative method for monitoring its regulation in hepatocytes and H4-II-E cells.
- Published
- 2007
- Full Text
- View/download PDF
25. Ames test-negative carcinogen, ortho-phenyl phenol, binds tubulin and causes aneuploidy in budding yeast.
- Author
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Nunoshiba T, Watanabe E, Takahashi T, Daigaku Y, Ishikawa S, Mochizuki M, Ui A, Enomoto T, and Yamamoto K
- Subjects
- Cell Division, Chromosomes, Fungal genetics, Flow Cytometry, G1 Phase, Loss of Heterozygosity, Microtubules metabolism, Aneuploidy, Biphenyl Compounds metabolism, Carcinogens metabolism, Hydroquinones metabolism, Saccharomyces cerevisiae genetics, Tubulin metabolism
- Abstract
Ortho-phenyl phenol (OPP) is broad-spectrum of fungicides and antibacterial agents. OPP tested negative in an Ames system and positive with respect to the formation of tumors in the urinary bladder in rats when administered in diet, showing attributes of an Ames test-negative carcinogen. It has also been demonstrated that OPP does not bind or cleave DNA in vivo or in vitro, rather dose-dependent protein binding in OPP-treated rats was observed. OPP, however, generates chromosomal aberrations including aneuploidy. Thus, the steps by which Ames test-negative carcinogens exert their effects need to be elucidated. Here, we used an assay of loss of heterozygosity (LOH) in Saccharomyces cerevisiae to determine the biological effects of OPP and its hepatic metabolite phenyl hydroquinone (PHQ). LOH was found to be induced by OPP and PHQ because of a functional chromosome loss: aneuploidy. PHQ bound to and interfered with the depolymerization of tubulin in vitro and arrested the cell-cycle at M and G1. These results indicate that OPP and PHQ damaged tubulin to cause mis-segregation of chromosome by delaying cell-cycle progression through mitosis, and as a consequence caused aneuploidy.
- Published
- 2007
- Full Text
- View/download PDF
26. Error-free RAD52 pathway and error-prone REV3 pathway determines spontaneous mutagenesis in Saccharomyces cerevisiae.
- Author
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Endo K, Tago Y, Daigaku Y, and Yamamoto K
- Subjects
- Amino Acid Transport Systems, Basic genetics, Amino Acid Transport Systems, Basic metabolism, DNA Mutational Analysis, DNA-Directed DNA Polymerase metabolism, Diploidy, Haploidy, Loss of Heterozygosity, Phenotype, Rad52 DNA Repair and Recombination Protein metabolism, Recombination, Genetic, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Signal Transduction genetics, DNA-Directed DNA Polymerase genetics, Mutagenesis physiology, Mutation, Rad52 DNA Repair and Recombination Protein genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics
- Abstract
Using the CAN1 gene in haploid cells or heterozygous diploid cells, we characterized the effects of mutations in the RAD52 and REV3 genes of Saccharomyces cerevisiae in spontaneous mutagenesis. The mutation rate was 5-fold higher in the haploid rad52 strain and 2.5-fold lower in rev3 than in the wild-type strain. The rate in the rad52 rev3 strain was as low as in the wild-type strain, indicating the rad52 mutator phenotype to be dependent on REV3. Sequencing indicated that G:C-->T:A and G:C-->C:G transversions increased in the rad52 strain and decreased in the rev3 and rad52 rev3 strains, suggesting a role for REV3 in transversion mutagenesis. In diploid rev3 cells, frequencies of can1Delta::LEU2/can1Delta::LEU2 from CAN1/can1Delta::LEU2 due to recombination were increased over the wild-type level. Overall, in the absence of RAD52, REV3-dependent base-substitutions increased, while in the absence of REV3, RAD52-dependent recombination events increased. We further found that the rad52 mutant had an increased rate of chromosome loss and the rad52 rev3 double mutant had an enhanced chromosome loss mutator phenotype. Taken together, our study indicates that the error-free RAD52 pathway and error-prone REV3 pathway for rescuing replication fork arrest determine spontaneous mutagenesis, recombination, and genome instability.
- Published
- 2007
- Full Text
- View/download PDF
27. Spontaneous mutagenesis in haploid and diploid Saccharomyces cerevisiae.
- Author
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Ohnishi G, Endo K, Doi A, Fujita A, Daigaku Y, Nunoshiba T, and Yamamoto K
- Subjects
- Chromosome Mapping methods, DNA Mutational Analysis methods, Gene Expression Regulation, Fungal genetics, Gene Frequency, Genetic Variation genetics, Amino Acid Transport Systems genetics, Chromosomes, Fungal genetics, Diploidy, Fungal Proteins genetics, Haploidy, Mutagenesis genetics, Saccharomyces cerevisiae genetics
- Abstract
To obtain insights into the mechanisms of spontaneous mutations in Saccharomyces cerevisiae, we have characterized the genetic alterations that inactivate either the CAN1 gene in haploid cells or heterozygously situated in diploid cells. The mutation rate in haploid cells was 9.08 x 10(-7), 100-fold lower than that in diploid cells (1.03 x 10(-4)). In haploid cells, among 69 independent CAN1 mutations, 75% were base substitutions and 22% frameshifts. The base substitutions were both transitions (33%) and transversions (42%), with G:C-->A:T and G:C-->T:A dominating. Minus frameshifts (12%) and plus frameshifts (10%) were also observed at run and non-run bases, and at A:T and G:C pairs with almost equal efficiency. An analysis of chromosome structure in diploid yeast cells indicated that allelic crossover was the predominant event followed by gene conversion and chromosome loss. We argued that genetic alterations leading to spontaneous phenotypic changes in wild-type diploid yeast cells occurred through two steps; replication-dependent alterations of bases in either allele then recombination-dependent transfer of the mutated allele to the intact one.
- Published
- 2004
- Full Text
- View/download PDF
28. Loss of heterozygosity and DNA damage repair in Saccharomyces cerevisiae.
- Author
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Daigaku Y, Endo K, Watanabe E, Ono T, and Yamamoto K
- Subjects
- Base Sequence, DNA Primers, DNA Damage, DNA Repair, Loss of Heterozygosity, Saccharomyces cerevisiae genetics
- Abstract
Loss of heterozygosity (LOH) of tumor suppressor genes is a crucial step in the development of sporadic and hereditary cancer. Understanding how LOH events arise may provide an opportunity for the prevention or early intervention of cancer development. In an effort to investigate the source of LOH events, we constructed MATalphacan1Delta::LEU2 and MATa CAN1 haploid yeast strains and examined canavanine-resistance mutations in a MATa CAN1/MATalphacan1Delta::LEU2 heterozygote formed by mating UV-irradiated and nonirradiated haploids. An increase in LOH was observed when the irradiated CAN1 haploid was mated with nonirradiated can1Delta::LEU2, while reversed irradiation only marginally increased LOH. In the rad51Delta background, allelic crossover type LOH increased following UV irradiation but not gene conversion. In the rad52Delta background, neither type of LOH increased. The chromosome structure following LOH and the requirement for Rad51 and Rad52 proteins indicated the involvement of gene conversion, allelic crossover and break-induced replication. We argued that LOH events could have occurred during the repair of double-strand breaks on a functional (damaged) but not nonfunctional (undamaged) chromosome through recombination.
- Published
- 2004
- Full Text
- View/download PDF
29. Saccharomyces cerevisiae RAD27 complements its Escherichia coli homolog in damage repair but not mutation avoidance.
- Author
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Ohnishi G, Daigaku Y, Nagata Y, Ihara M, and Yamamoto K
- Subjects
- Acetyltransferases, DNA Polymerase I genetics, DNA Polymerase I metabolism, Escherichia coli genetics, Escherichia coli Proteins genetics, Flap Endonucleases genetics, Genetic Complementation Test, Membrane Proteins genetics, Membrane Proteins metabolism, Mutation, Promoter Regions, Genetic, Saccharomyces cerevisiae Proteins genetics, DNA Repair genetics, Escherichia coli Proteins metabolism, Flap Endonucleases metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
In eukaryotes, the flap endonuclease of Rad27/Fen-1 is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5' ends of Okazaki fragments, and in base excision repair by cleaving a 5' flap structure that may result during base excision repair. Saccharomyces cerevisiae rad27Delta mutants further display a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result from duplications of DNA sequence, indicating a role in mutation avoidance. Two conserved motifs in Rad27/Fen-1 show homology to the 5' --> 3' exonuclease domain of Escherichia coli DNA polymerase I. The strain defective in the 5' --> 3' exonuclease domain in DNA polymerase I shows essentially the same phenotype as the yeast rad27Delta strain. In this study, we expressed the yeast RAD27 gene in an E. coli strain lacking the 5' --> 3' exonuclease domain in DNA polymerase I in order to test whether eukaryotic RAD27/FEN-1 can complement the defect of its bacterial homolog. We found that the yeast Rad27 protein complements sensitivity to methyl methanesulfonate in an E. coli mutant. On the other hand, Rad27 protein did not reduce the high rate of spontaneous mutagenesis in the E. coli tonB gene which results from duplication of DNA. These results indicate that the yeast Rad27 and E. coli 5' --> 3' exonuclease act on the same substrate. We argue that the lack of mutation avoidance of yeast RAD27 in E. coli results from a lack of interaction between the yeast Rad27 protein and the E. coli replication clamp (beta-clamp).
- Published
- 2004
- Full Text
- View/download PDF
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