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Yeast require redox switching in DNA primase.
O'Brien E, Salay LE, Epum EA, Friedman KL, Chazin WJ, Barton JK
(2018) Proc Natl Acad Sci U S A 115: 13186-13191
MeSH Terms: Crystallography, X-Ray, DNA Primase, Electron Transport, Iron-Sulfur Proteins, Models, Molecular, Mutation, Oxidation-Reduction, Protein Conformation, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Show Abstract · Added March 26, 2019
Eukaryotic DNA primases contain a [4Fe4S] cluster in the C-terminal domain of the p58 subunit (p58C) that affects substrate affinity but is not required for catalysis. We show that, in yeast primase, the cluster serves as a DNA-mediated redox switch governing DNA binding, just as in human primase. Despite a different structural arrangement of tyrosines to facilitate electron transfer between the DNA substrate and [4Fe4S] cluster, in yeast, mutation of tyrosines Y395 and Y397 alters the same electron transfer chemistry and redox switch. Mutation of conserved tyrosine 395 diminishes the extent of p58C participation in normal redox-switching reactions, whereas mutation of conserved tyrosine 397 causes oxidative cluster degradation to the [3Fe4S] species during p58C redox signaling. Switching between oxidized and reduced states in the presence of the Y397 mutations thus puts primase [4Fe4S] cluster integrity and function at risk. Consistent with these observations, we find that yeast tolerate mutations to Y395 in p58C, but the single-residue mutation Y397L in p58C is lethal. Our data thus show that a constellation of tyrosines for protein-DNA electron transfer mediates the redox switch in eukaryotic primases and is required for primase function in vivo.
Copyright © 2018 the Author(s). Published by PNAS.
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MeSH Terms
Ubiquitin turnover and endocytic trafficking in yeast are regulated by Ser57 phosphorylation of ubiquitin.
Lee S, Tumolo JM, Ehlinger AC, Jernigan KK, Qualls-Histed SJ, Hsu PC, McDonald WH, Chazin WJ, MacGurn JA
(2017) Elife 6:
MeSH Terms: Endocytosis, Homeostasis, Phosphoprotein Phosphatases, Phosphorylation, Protein Processing, Post-Translational, Saccharomyces cerevisiae Proteins, Ubiquitin, Yeasts
Show Abstract · Added March 24, 2018
Despite its central role in protein degradation little is known about the molecular mechanisms that sense, maintain, and regulate steady state concentration of ubiquitin in the cell. Here, we describe a novel mechanism for regulation of ubiquitin homeostasis that is mediated by phosphorylation of ubiquitin at the Ser57 position. We find that loss of Ppz phosphatase activity leads to defects in ubiquitin homeostasis that are at least partially attributable to elevated levels of Ser57 phosphorylated ubiquitin. Phosphomimetic mutation at the Ser57 position of ubiquitin conferred increased rates of endocytic trafficking and ubiquitin turnover. These phenotypes are associated with bypass of recognition by endosome-localized deubiquitylases - including Doa4 which is critical for regulation of ubiquitin recycling. Thus, ubiquitin homeostasis is significantly impacted by the rate of ubiquitin flux through the endocytic pathway and by signaling pathways that converge on ubiquitin itself to determine whether it is recycled or degraded in the vacuole.
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8 MeSH Terms
COPI mediates recycling of an exocytic SNARE by recognition of a ubiquitin sorting signal.
Xu P, Hankins HM, MacDonald C, Erlinger SJ, Frazier MN, Diab NS, Piper RC, Jackson LP, MacGurn JA, Graham TR
(2017) Elife 6:
MeSH Terms: Coat Protein Complex I, Exosomes, Protein Transport, R-SNARE Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Ubiquitin
Show Abstract · Added March 14, 2018
The COPI coat forms transport vesicles from the Golgi complex and plays a poorly defined role in endocytic trafficking. Here we show that COPI binds K63-linked polyubiquitin and this interaction is crucial for trafficking of a ubiquitinated yeast SNARE (Snc1). Snc1 is a v-SNARE that drives fusion of exocytic vesicles with the plasma membrane, and then recycles through the endocytic pathway to the Golgi for reuse in exocytosis. Removal of ubiquitin from Snc1, or deletion of a β'-COP subunit propeller domain that binds K63-linked polyubiquitin, disrupts Snc1 recycling causing aberrant accumulation in internal compartments. Moreover, replacement of the β'-COP propeller domain with unrelated ubiquitin-binding domains restores Snc1 recycling. These results indicate that ubiquitination, a modification well known to target membrane proteins to the lysosome or vacuole for degradation, can also function as recycling signal to sort a SNARE into COPI vesicles in a non-degradative pathway.
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7 MeSH Terms
Nup42 and IP coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells.
Adams RL, Mason AC, Glass L, Aditi , Wente SR
(2017) Traffic 18: 776-790
MeSH Terms: Active Transport, Cell Nucleus, DEAD-box RNA Helicases, Humans, Nuclear Pore, Nuclear Pore Complex Proteins, Nucleocytoplasmic Transport Proteins, Phytic Acid, RNA, Messenger, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Show Abstract · Added March 30, 2018
The mRNA lifecycle is driven through spatiotemporal changes in the protein composition of mRNA particles (mRNPs) that are triggered by RNA-dependent DEAD-box protein (Dbp) ATPases. As mRNPs exit the nuclear pore complex (NPC) in Saccharomyces cerevisiae, this remodeling occurs through activation of Dbp5 by inositol hexakisphosphate (IP )-bound Gle1. At the NPC, Gle1 also binds Nup42, but Nup42's molecular function is unclear. Here we employ the power of structure-function analysis in S. cerevisiae and human (h) cells, and find that the high-affinity Nup42-Gle1 interaction is integral to Dbp5 (hDDX19B) activation and efficient mRNA export. The Nup42 carboxy-terminal domain (CTD) binds Gle1/hGle1B at an interface distinct from the Gle1-Dbp5/hDDX19B interaction site. A nup42-CTD/gle1-CTD/Dbp5 trimeric complex forms in the presence of IP . Deletion of NUP42 abrogates Gle1-Dbp5 interaction, and disruption of the Nup42 or IP binding interfaces on Gle1/hGle1B leads to defective mRNA export in S. cerevisiae and human cells. In vitro, Nup42-CTD and IP stimulate Gle1/hGle1B activation of Dbp5 and DDX19B recombinant proteins in similar, nonadditive manners, demonstrating complete functional conservation between humans and S. cerevisiae. Together, a highly conserved mechanism governs spatial coordination of mRNP remodeling during export. This has implications for understanding human disease mutations that perturb the Nup42-hGle1B interaction.
© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
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10 MeSH Terms
Analysis of DNA binding by human factor xeroderma pigmentosum complementation group A (XPA) provides insight into its interactions with nucleotide excision repair substrates.
Sugitani N, Voehler MW, Roh MS, Topolska-Woś AM, Chazin WJ
(2017) J Biol Chem 292: 16847-16857
MeSH Terms: Amino Acid Substitution, DNA Repair, DNA Repair Enzymes, DNA, Single-Stranded, Humans, Mutation, Missense, Nuclear Magnetic Resonance, Biomolecular, Protein Binding, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Structural Homology, Protein, Xeroderma Pigmentosum, Xeroderma Pigmentosum Group A Protein
Show Abstract · Added March 24, 2018
Xeroderma pigmentosum (XP) complementation group A (XPA) is an essential scaffolding protein in the multiprotein nucleotide excision repair (NER) machinery. The interaction of XPA with DNA is a core function of this protein; a number of mutations in the DNA-binding domain (DBD) are associated with XP disease. Although structures of the central globular domain of human XPA and data on binding of DNA substrates have been reported, the structural basis for XPA's DNA-binding activity remains unknown. X-ray crystal structures of the central globular domain of yeast XPA (Rad14) with lesion-containing DNA duplexes have provided valuable insights, but the DNA substrates used for this study do not correspond to the substrates of XPA as it functions within the NER machinery. To better understand the DNA-binding activity of human XPA in NER, we used NMR to investigate the interaction of its DBD with a range of DNA substrates. We found that XPA binds different single-stranded/double-stranded junction DNA substrates with a common surface. Comparisons of our NMR-based mapping of binding residues with the previously reported Rad14-DNA crystal structures revealed similarities and differences in substrate binding between XPA and Rad14. This includes direct evidence for DNA contacts to the residues extending C-terminally from the globular core, which are lacking in the Rad14 construct. Moreover, mutation of the XPA residue corresponding to Phe-262 in Rad14, previously reported as being critical for DNA binding, had only a moderate effect on the DNA-binding activity of XPA. The DNA-binding properties of several disease-associated mutations in the DBD were investigated. These results suggest that for XPA mutants exhibiting altered DNA-binding properties, a correlation exists between the extent of reduction in DNA-binding affinity and the severity of symptoms in XP patients.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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13 MeSH Terms
Mms1 binds to G-rich regions in Saccharomyces cerevisiae and influences replication and genome stability.
Wanzek K, Schwindt E, Capra JA, Paeschke K
(2017) Nucleic Acids Res 45: 7796-7806
MeSH Terms: Binding Sites, Cell Cycle, Cullin Proteins, DNA Helicases, DNA Replication, DNA, Fungal, Endodeoxyribonucleases, Exodeoxyribonucleases, G-Quadruplexes, GC Rich Sequence, Genomic Instability, Models, Biological, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins
Show Abstract · Added March 14, 2018
The regulation of replication is essential to preserve genome integrity. Mms1 is part of the E3 ubiquitin ligase complex that is linked to replication fork progression. By identifying Mms1 binding sites genome-wide in Saccharomyces cerevisiae we connected Mms1 function to genome integrity and replication fork progression at particular G-rich motifs. This motif can form G-quadruplex (G4) structures in vitro. G4 are stable DNA structures that are known to impede replication fork progression. In the absence of Mms1, genome stability is at risk at these G-rich/G4 regions as demonstrated by gross chromosomal rearrangement assays. Mms1 binds throughout the cell cycle to these G-rich/G4 regions and supports the binding of Pif1 DNA helicase. Based on these data we propose a mechanistic model in which Mms1 binds to specific G-rich/G4 motif located on the lagging strand template for DNA replication and supports Pif1 function, DNA replication and genome integrity.
© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.
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14 MeSH Terms
Extensive Copy Number Variation in Fermentation-Related Genes Among Wine Strains.
Steenwyk J, Rokas A
(2017) G3 (Bethesda) 7: 1475-1485
MeSH Terms: DNA Copy Number Variations, Fermentation, Genes, Fungal, Saccharomyces cerevisiae, Wine
Show Abstract · Added April 6, 2017
Due to the importance of in wine-making, the genomic variation of wine yeast strains has been extensively studied. One of the major insights stemming from these studies is that wine yeast strains harbor low levels of genetic diversity in the form of single nucleotide polymorphisms (SNPs). Genomic structural variants, such as copy number (CN) variants, are another major type of variation segregating in natural populations. To test whether genetic diversity in CN variation is also low across wine yeast strains, we examined genome-wide levels of CN variation in 132 whole-genome sequences of wine strains. We found an average of 97.8 CN variable regions (CNVRs) affecting ∼4% of the genome per strain. Using two different measures of CN diversity, we found that gene families involved in fermentation-related processes such as copper resistance (), flocculation (), and glucose metabolism (), as well as the gene family whose members are expressed before or during the diauxic shift, showed substantial CN diversity across the 132 strains examined. Importantly, these same gene families have been shown, through comparative transcriptomic and functional assays, to be associated with adaptation to the wine fermentation environment. Our results suggest that CN variation is a substantial contributor to the genomic diversity of wine yeast strains, and identify several candidate loci whose levels of CN variation may affect the adaptation and performance of wine yeast strains during fermentation.
Copyright © 2017 Steenwyk and Rokas.
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5 MeSH Terms
Nup100 regulates replicative life span by mediating the nuclear export of specific tRNAs.
Lord CL, Ospovat O, Wente SR
(2017) RNA 23: 365-377
MeSH Terms: Active Transport, Cell Nucleus, Basic-Leucine Zipper Transcription Factors, Blotting, Northern, Cell Division, Cell Nucleus, Culture Media, Gene Expression Regulation, Fungal, In Situ Hybridization, Fluorescence, Karyopherins, Nuclear Pore, Nuclear Pore Complex Proteins, RNA, Fungal, RNA, Transfer, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Time Factors
Show Abstract · Added April 14, 2017
Nuclear pore complexes (NPCs), which are composed of nucleoporins (Nups) and regulate transport between the nucleus and cytoplasm, significantly impact the replicative life span (RLS) of We previously reported that deletion of the nonessential gene increases RLS, although the molecular basis for this effect was unknown. In this study, we find that nuclear tRNA accumulation contributes to increased longevity in Δ cells. Fluorescence in situ hybridization (FISH) experiments demonstrate that several specific tRNAs accumulate in the nuclei of Δ mutants. Protein levels of the transcription factor Gcn4 are increased when is deleted, and is required for the elevated life spans of Δ mutants, similar to other previously described tRNA export and ribosomal mutants. Northern blots indicate that tRNA splicing and aminoacylation are not significantly affected in Δ cells, suggesting that Nup100 is largely required for nuclear export of mature, processed tRNAs. Distinct tRNAs accumulate in the nuclei of Δ and Δ mutants, while Los1-GFP nucleocytoplasmic shuttling is unaffected by Nup100. Thus, we conclude that Nup100 regulates tRNA export in a manner distinct from Los1 or Msn5. Together, these experiments reveal a novel Nup100 role in the tRNA life cycle that impacts the life span.
© 2017 Lord et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.
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16 MeSH Terms
Trapping redox partnerships in oxidant-sensitive proteins with a small, thiol-reactive cross-linker.
Allan KM, Loberg MA, Chepngeno J, Hurtig JE, Tripathi S, Kang MG, Allotey JK, Widdershins AH, Pilat JM, Sizek HJ, Murphy WJ, Naticchia MR, David JB, Morano KA, West JD
(2016) Free Radic Biol Med 101: 356-366
MeSH Terms: Cross-Linking Reagents, Disulfides, Glutathione Peroxidase, Methionine Sulfoxide Reductases, Oxidants, Oxidation-Reduction, Oxidative Stress, Oxidoreductases Acting on Sulfur Group Donors, Peroxiredoxins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Sulfhydryl Compounds, Sulfones, Thioredoxins, tert-Butylhydroperoxide
Show Abstract · Added April 24, 2017
A broad range of redox-regulated proteins undergo reversible disulfide bond formation on oxidation-prone cysteine residues. Heightened reactivity of the thiol groups in these cysteines also increases susceptibility to modification by organic electrophiles, a property that can be exploited in the study of redox networks. Here, we explored whether divinyl sulfone (DVSF), a thiol-reactive bifunctional electrophile, cross-links oxidant-sensitive proteins to their putative redox partners in cells. To test this idea, previously identified oxidant targets involved in oxidant defense (namely, peroxiredoxins, methionine sulfoxide reductases, sulfiredoxin, and glutathione peroxidases), metabolism, and proteostasis were monitored for cross-link formation following treatment of Saccharomyces cerevisiae with DVSF. Several proteins screened, including multiple oxidant defense proteins, underwent intermolecular and/or intramolecular cross-linking in response to DVSF. Specific redox-active cysteines within a subset of DVSF targets were found to influence cross-linking; in addition, DVSF-mediated cross-linking of its targets was impaired in cells first exposed to oxidants. Since cross-linking appeared to involve redox-active cysteines in these proteins, we examined whether potential redox partners became cross-linked to them upon DVSF treatment. Specifically, we found that several substrates of thioredoxins were cross-linked to the cytosolic thioredoxin Trx2 in cells treated with DVSF. However, other DVSF targets, like the peroxiredoxin Ahp1, principally formed intra-protein cross-links upon DVSF treatment. Moreover, additional protein targets, including several known to undergo S-glutathionylation, were conjugated via DVSF to glutathione. Our results indicate that DVSF is of potential use as a chemical tool for irreversibly trapping and discovering thiol-based redox partnerships within cells.
Copyright © 2016 Elsevier Inc. All rights reserved.
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15 MeSH Terms
Exploring genetic suppression interactions on a global scale.
van Leeuwen J, Pons C, Mellor JC, Yamaguchi TN, Friesen H, Koschwanez J, Ušaj MM, Pechlaner M, Takar M, Ušaj M, VanderSluis B, Andrusiak K, Bansal P, Baryshnikova A, Boone CE, Cao J, Cote A, Gebbia M, Horecka G, Horecka I, Kuzmin E, Legro N, Liang W, van Lieshout N, McNee M, San Luis BJ, Shaeri F, Shuteriqi E, Sun S, Yang L, Youn JY, Yuen M, Costanzo M, Gingras AC, Aloy P, Oostenbrink C, Murray A, Graham TR, Myers CL, Andrews BJ, Roth FP, Boone C
(2016) Science 354:
MeSH Terms: Cell Physiological Phenomena, Chromosome Mapping, Gene Regulatory Networks, Genes, Fungal, Genes, Suppressor, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Suppression, Genetic
Show Abstract · Added April 6, 2017
Genetic suppression occurs when the phenotypic defects caused by a mutation in a particular gene are rescued by a mutation in a second gene. To explore the principles of genetic suppression, we examined both literature-curated and unbiased experimental data, involving systematic genetic mapping and whole-genome sequencing, to generate a large-scale suppression network among yeast genes. Most suppression pairs identified novel relationships among functionally related genes, providing new insights into the functional wiring diagram of the cell. In addition to suppressor mutations, we identified frequent secondary mutations,in a subset of genes, that likely cause a delay in the onset of stationary phase, which appears to promote their enrichment within a propagating population. These findings allow us to formulate and quantify general mechanisms of genetic suppression.
Copyright © 2016, American Association for the Advancement of Science.
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8 MeSH Terms