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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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Substrate Binding Regulates Redox Signaling in Human DNA Primase.
O'Brien E, Holt ME, Salay LE, Chazin WJ, Barton JK
(2018) J Am Chem Soc 140: 17153-17162
MeSH Terms: DNA, DNA Primase, Electrochemical Techniques, Humans, Iron-Sulfur Proteins, Nucleotides, Oxidation-Reduction, Protein Binding, Protein Domains, Transcription Elongation, Genetic, Transcription Initiation, Genetic
Show Abstract · Added March 26, 2019
Generation of daughter strands during DNA replication requires the action of DNA primase to synthesize an initial short RNA primer on the single-stranded DNA template. Primase is a heterodimeric enzyme containing two domains whose activity must be coordinated during primer synthesis: an RNA polymerase domain in the small subunit (p48) and a [4Fe4S] cluster-containing C-terminal domain of the large subunit (p58C). Here we examine the redox switching properties of the [4Fe4S] cluster in the full p48/p58 heterodimer using DNA electrochemistry. Unlike with isolated p58C, robust redox signaling in the primase heterodimer requires binding of both DNA and NTPs; NTP binding shifts the p48/p58 cluster redox potential into the physiological range, generating a signal near 160 mV vs NHE. Preloading of primase with NTPs enhances catalytic activity on primed DNA, suggesting that primase configurations promoting activity are more highly populated in the NTP-bound protein. We propose that p48/p58 binding of anionic DNA and NTPs affects the redox properties of the [4Fe4S] cluster; this electrostatic change is likely influenced by the alignment of primase subunits during activity because the configuration affects the [4Fe4S] cluster environment and coupling to DNA bases for redox signaling. Thus, both binding of polyanionic substrates and configurational dynamics appear to influence [4Fe4S] redox signaling properties. These results suggest that these factors should be considered generally in characterizing signaling networks of large, multisubunit DNA-processing [4Fe4S] enzymes.
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11 MeSH Terms
A Polymerase With Potential: The Fe-S Cluster in Human DNA Primase.
Holt ME, Salay LE, Chazin WJ
(2017) Methods Enzymol 595: 361-390
MeSH Terms: Catalytic Domain, DNA, DNA Primase, DNA Primers, DNA Replication, Humans, Iron-Sulfur Proteins, Oxidation-Reduction, RNA, Sequence Analysis
Show Abstract · Added March 24, 2018
Replication of DNA in eukaryotes is primarily executed by the combined action of processive DNA polymerases δ and ɛ. These enzymes cannot initiate synthesis of new DNA without the presence of a primer on the template ssDNA. The primers on both the leading and lagging strands are generated by DNA polymerase α-primase (pol-prim). DNA primase is a DNA-dependent RNA polymerase that synthesizes the first ~10 nucleotides and then transfers the substrate to polymerase α to complete primer synthesis. The mechanisms governing the coordination and handoff between primase and polymerase α are largely unknown. Isolated DNA primase contains a [4Fe-4S] cluster that has been shown to serve as a redox switch modulating DNA binding affinity. This discovery suggests a mechanism for modulating the priming activity of primase and handoff to polymerase α. In this chapter, we briefly discuss the current state of knowledge of primase structure and function, including the role of its iron-sulfur cluster. This is followed by providing the methods for expressing, purifying, and biophysically/structurally characterizing primase and its iron-sulfur cluster-containing domain, p58C.
© 2017 Elsevier Inc. All rights reserved.
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10 MeSH Terms
Hydrogen Sulfide and Reactive Sulfur Species Impact Proteome S-Sulfhydration and Global Virulence Regulation in Staphylococcus aureus.
Peng H, Zhang Y, Palmer LD, Kehl-Fie TE, Skaar EP, Trinidad JC, Giedroc DP
(2017) ACS Infect Dis 3: 744-755
MeSH Terms: Gene Expression Regulation, Bacterial, Hydrogen Sulfide, Proteome, Staphylococcus aureus, Sulfur, Virulence
Show Abstract · Added September 23, 2017
Hydrogen sulfide (HS) is thought to protect bacteria from oxidative stress, but a comprehensive understanding of its function in bacteria is largely unexplored. In this study, we show that the human pathogen Staphylococcus aureus (S. aureus) harbors significant effector molecules of HS signaling, reactive sulfur species (RSS), as low molecular weight persulfides of bacillithiol, coenzyme A, and cysteine, and significant inorganic polysulfide species. We find that proteome S-sulfhydration, a post-translational modification (PTM) in HS signaling, is widespread in S. aureus. RSS levels modulate the expression of secreted virulence factors and the cytotoxicity of the secretome, consistent with an S-sulfhydration-dependent inhibition of DNA binding by MgrA, a global virulence regulator. Two previously uncharacterized thioredoxin-like proteins, denoted TrxP and TrxQ, are S-sulfhydrated in sulfide-stressed cells and are capable of reducing protein hydrodisulfides, suggesting that this PTM is potentially regulatory in S. aureus. In conclusion, our results reveal that S. aureus harbors a pool of proteome- and metabolite-derived RSS capable of impacting protein activities and gene regulation and that HS signaling can be sensed by global regulators to affect the expression of virulence factors.
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6 MeSH Terms
Introduction to Metals in Biology 2017: Iron transport, storage, and the ramifications.
Guengerich FP
(2017) J Biol Chem 292: 12725-12726
MeSH Terms: Animals, Biological Transport, Homeostasis, Humans, Iron, Iron-Regulatory Proteins, Iron-Sulfur Proteins
Show Abstract · Added March 14, 2018
In this tenth Thematic Series in Metals in Biology, six Minireviews deal with aspects of iron metabolism. A number of important proteins control iron homeostasis, including hepcidin and ferroportin, in various cells. Other aspects of iron dealt with here include biogenesis of iron-sulfur proteins and chaperones that deliver iron cofactors in cells. Additionally, an iron-regulated metastasis suppressor interacts with the epidermal growth factor receptor and mediates its downstream signaling activity.
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
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7 MeSH Terms
The [4Fe4S] cluster of human DNA primase functions as a redox switch using DNA charge transport.
O'Brien E, Holt ME, Thompson MK, Salay LE, Ehlinger AC, Chazin WJ, Barton JK
(2017) Science 355:
MeSH Terms: Biological Transport, DNA, DNA Primase, DNA Replication, Electrolysis, Humans, Iron-Sulfur Proteins, Mutation, Oxidation-Reduction, Polymerization, Protein Binding, Protein Domains
Show Abstract · Added March 24, 2018
DNA charge transport chemistry offers a means of long-range, rapid redox signaling. We demonstrate that the [4Fe4S] cluster in human DNA primase can make use of this chemistry to coordinate the first steps of DNA synthesis. Using DNA electrochemistry, we found that a change in oxidation state of the [4Fe4S] cluster acts as a switch for DNA binding. Single-atom mutations that inhibit this charge transfer hinder primase initiation without affecting primase structure or polymerization. Generating a single base mismatch in the growing primer duplex, which attenuates DNA charge transport, inhibits primer truncation. Thus, redox signaling by [4Fe4S] clusters using DNA charge transport regulates primase binding to DNA and illustrates chemistry that may efficiently drive substrate handoff between polymerases during DNA replication.
Copyright © 2017, American Association for the Advancement of Science.
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12 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
A Small-Molecule Inhibitor of Iron-Sulfur Cluster Assembly Uncovers a Link between Virulence Regulation and Metabolism in Staphylococcus aureus.
Choby JE, Mike LA, Mashruwala AA, Dutter BF, Dunman PM, Sulikowski GA, Boyd JM, Skaar EP
(2016) Cell Chem Biol 23: 1351-1361
MeSH Terms: Aconitate Hydratase, Anti-Bacterial Agents, Bacterial Proteins, Drug Discovery, Humans, Iron-Sulfur Proteins, Protein Kinases, Signal Transduction, Small Molecule Libraries, Staphylococcal Infections, Staphylococcus aureus, Transcription Factors, Virulence, Virulence Factors
Show Abstract · Added April 8, 2017
The rising problem of antimicrobial resistance in Staphylococcus aureus necessitates the discovery of novel therapeutic targets for small-molecule intervention. A major obstacle of drug discovery is identifying the target of molecules selected from high-throughput phenotypic assays. Here, we show that the toxicity of a small molecule termed '882 is dependent on the constitutive activity of the S. aureus virulence regulator SaeRS, uncovering a link between virulence factor production and energy generation. A series of genetic, physiological, and biochemical analyses reveal that '882 inhibits iron-sulfur (Fe-S) cluster assembly most likely through inhibition of the Suf complex, which synthesizes Fe-S clusters. In support of this, '882 supplementation results in decreased activity of the Fe-S cluster-dependent enzyme aconitase. Further information regarding the effects of '882 has deepened our understanding of virulence regulation and demonstrates the potential for small-molecule modulation of Fe-S cluster assembly in S. aureus and other pathogens.
Copyright © 2016 Elsevier Ltd. All rights reserved.
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14 MeSH Terms
Bacillithiol has a role in Fe-S cluster biogenesis in Staphylococcus aureus.
Rosario-Cruz Z, Chahal HK, Mike LA, Skaar EP, Boyd JM
(2015) Mol Microbiol 98: 218-42
MeSH Terms: Aconitate Hydratase, Apoproteins, Bacterial Proteins, Cysteine, Glucosamine, Glutamate Synthase, Homeostasis, Iron, Iron-Sulfur Proteins, Oxidation-Reduction, Phenotype, Staphylococcus aureus, Sulfur
Show Abstract · Added February 8, 2016
Staphylococcus aureus does not produce the low-molecular-weight (LMW) thiol glutathione, but it does produce the LMW thiol bacillithiol (BSH). To better understand the roles that BSH plays in staphylococcal metabolism, we constructed and examined strains lacking BSH. Phenotypic analysis found that the BSH-deficient strains cultured either aerobically or anaerobically had growth defects that were alleviated by the addition of exogenous iron (Fe) or the amino acids leucine and isoleucine. The activities of the iron-sulfur (Fe-S) cluster-dependent enzymes LeuCD and IlvD, which are required for the biosynthesis of leucine and isoleucine, were decreased in strains lacking BSH. The BSH-deficient cells also had decreased aconitase and glutamate synthase activities, suggesting a general defect in Fe-S cluster biogenesis. The phenotypes of the BSH-deficient strains were exacerbated in strains lacking the Fe-S cluster carrier Nfu and partially suppressed by multicopy expression of either sufA or nfu, suggesting functional overlap between BSH and Fe-S carrier proteins. Biochemical analysis found that SufA bound and transferred Fe-S clusters to apo-aconitase, verifying that it serves as an Fe-S cluster carrier. The results presented are consistent with the hypothesis that BSH has roles in Fe homeostasis and the carriage of Fe-S clusters to apo-proteins in S. aureus.
© 2015 John Wiley & Sons Ltd.
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13 MeSH Terms
Nfu facilitates the maturation of iron-sulfur proteins and participates in virulence in Staphylococcus aureus.
Mashruwala AA, Pang YY, Rosario-Cruz Z, Chahal HK, Benson MA, Mike LA, Skaar EP, Torres VJ, Nauseef WM, Boyd JM
(2015) Mol Microbiol 95: 383-409
MeSH Terms: Aconitate Hydratase, Animals, DNA Damage, Disease Models, Animal, Humans, Iron, Iron-Sulfur Proteins, Mice, Multigene Family, Mutation, Neutrophils, Oxidation-Reduction, Protein Binding, Reactive Nitrogen Species, Reactive Oxygen Species, Staphylococcal Infections, Staphylococcus aureus, Sulfur, Virulence
Show Abstract · Added January 24, 2015
The acquisition and metabolism of iron (Fe) by the human pathogen Staphylococcus aureus is critical for disease progression. S. aureus requires Fe to synthesize inorganic cofactors called iron-sulfur (Fe-S) clusters, which are required for functional Fe-S proteins. In this study we investigated the mechanisms utilized by S. aureus to metabolize Fe-S clusters. We identified that S. aureus utilizes the Suf biosynthetic system to synthesize Fe-S clusters and we provide genetic evidence suggesting that the sufU and sufB gene products are essential. Additional biochemical and genetic analyses identified Nfu as an Fe-S cluster carrier, which aids in the maturation of Fe-S proteins. We find that deletion of the nfu gene negatively impacts staphylococcal physiology and pathogenicity. A nfu mutant accumulates both increased intracellular non-incorporated Fe and endogenous reactive oxygen species (ROS) resulting in DNA damage. In addition, a strain lacking Nfu is sensitive to exogenously supplied ROS and reactive nitrogen species. Congruous with ex vivo findings, a nfu mutant strain is more susceptible to oxidative killing by human polymorphonuclear leukocytes and displays decreased tissue colonization in a murine model of infection. We conclude that Nfu is necessary for staphylococcal pathogenesis and establish Fe-S cluster metabolism as an attractive antimicrobial target.
© 2014 John Wiley & Sons Ltd.
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19 MeSH Terms