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2.
Nat Commun ; 13(1): 5500, 2022 09 20.
Article de Anglais | MEDLINE | ID: mdl-36127359

RÉSUMÉ

Insulin-like growth factor (IGF) signaling is highly conserved and tightly regulated by proteases including Pregnancy-Associated Plasma Protein A (PAPP-A). PAPP-A and its paralog PAPP-A2 are metalloproteases that mediate IGF bioavailability through cleavage of IGF binding proteins (IGFBPs). Here, we present single-particle cryo-EM structures of the catalytically inactive mutant PAPP-A (E483A) in complex with a peptide from its substrate IGFBP5 (PAPP-ABP5) and also in its substrate-free form, by leveraging the power of AlphaFold to generate a high quality predicted model as a starting template. We show that PAPP-A is a flexible trans-dimer that binds IGFBP5 via a 25-amino acid anchor peptide which extends into the metalloprotease active site. This unique IGFBP5 anchor peptide that mediates the specific PAPP-A-IGFBP5 interaction is not found in other PAPP-A substrates. Additionally, we illustrate the critical role of the PAPP-A central domain as it mediates both IGFBP5 recognition and trans-dimerization. We further demonstrate that PAPP-A trans-dimer formation and distal inter-domain interactions are both required for efficient proteolysis of IGFBP4, but dispensable for IGFBP5 cleavage. Together the structural and biochemical studies reveal the mechanism of PAPP-A substrate binding and selectivity.


Sujet(s)
Protéine A plasmatique associée à la grossesse , Somatomédines , Acides aminés/métabolisme , Peptides/métabolisme , Protéine A plasmatique associée à la grossesse/composition chimique , Protéine A plasmatique associée à la grossesse/métabolisme , Liaison aux protéines , Somatomédines/métabolisme
3.
Sci Transl Med ; 13(613): eabj1578, 2021 Sep 29.
Article de Anglais | MEDLINE | ID: mdl-34586829

RÉSUMÉ

Interactions between WD40 repeat domain protein 5 (WDR5) and its various partners such as mixed lineage leukemia (MLL) and c-MYC are essential for sustaining oncogenesis in human cancers. However, inhibitors that block protein-protein interactions (PPIs) between WDR5 and its binding partners exhibit modest cancer cell killing effects and lack in vivo efficacy. Here, we present pharmacological degradation of WDR5 as a promising therapeutic strategy for treating WDR5-dependent tumors and report two high-resolution crystal structures of WDR5-degrader-E3 ligase ternary complexes. We identified an effective WDR5 degrader via structure-based design and demonstrated its in vitro and in vivo antitumor activities. On the basis of the crystal structure of an initial WDR5 degrader in complex with WDR5 and the E3 ligase von Hippel­Lindau (VHL), we designed a WDR5 degrader, MS67, and demonstrated the high cooperativity of MS67 binding to WDR5 and VHL by another ternary complex structure and biophysical characterization. MS67 potently and selectively depleted WDR5 and was more effective than WDR5 PPI inhibitors in suppressing transcription of WDR5-regulated genes, decreasing the chromatin-bound fraction of MLL complex components and c-MYC, and inhibiting the proliferation of cancer cells. In addition, MS67 suppressed malignant growth of MLL-rearranged acute myeloid leukemia patient cells in vitro and in vivo and was well tolerated in vivo. Collectively, our results demonstrate that structure-based design can be an effective strategy to identify highly active degraders and suggest that pharmacological degradation of WDR5 might be a promising treatment for WDR5-dependent cancers.


Sujet(s)
Leucémie aigüe myéloïde , Protéine de la leucémie myéloïde-lymphoïde , Animaux , Histone-lysine N-methyltransferase , Humains , Protéines et peptides de signalisation intracellulaire , Leucémie aigüe myéloïde/traitement médicamenteux , Leucémie aigüe myéloïde/génétique , Souris
4.
Nat Commun ; 12(1): 3440, 2021 06 08.
Article de Anglais | MEDLINE | ID: mdl-34103529

RÉSUMÉ

The multi-subunit translation initiation factor eIF2B is a control node for protein synthesis. eIF2B activity is canonically modulated through stress-responsive phosphorylation of its substrate eIF2. The eIF2B regulatory subcomplex is evolutionarily related to sugar-metabolizing enzymes, but the biological relevance of this relationship was unknown. To identify natural ligands that might regulate eIF2B, we conduct unbiased binding- and activity-based screens followed by structural studies. We find that sugar phosphates occupy the ancestral catalytic site in the eIF2Bα subunit, promote eIF2B holoenzyme formation and enhance enzymatic activity towards eIF2. A mutant in the eIF2Bα ligand pocket that causes Vanishing White Matter disease fails to engage and is not stimulated by sugar phosphates. These data underscore the importance of allosteric metabolite modulation for proper eIF2B function. We propose that eIF2B evolved to couple nutrient status via sugar phosphate sensing with the rate of protein synthesis, one of the most energetically costly cellular processes.


Sujet(s)
Facteur-2B d'initiation eucaryote/métabolisme , Stress physiologique , Oses phosphates/métabolisme , Régulation allostérique , Sites de fixation , Séquence conservée , Cryomicroscopie électronique , Facteur-2B d'initiation eucaryote/composition chimique , Facteur-2B d'initiation eucaryote/ultrastructure , Évolution moléculaire , Guanosine diphosphate/métabolisme , Cellules HEK293 , Humains , Leucoencéphalopathies/anatomopathologie , Ligands , Métabolome , Modèles moléculaires , Mutation/génétique , Sous-unités de protéines/composition chimique , Sous-unités de protéines/métabolisme , Spécificité du substrat , Oses phosphates/composition chimique
5.
Clin Cancer Res ; 27(16): 4652-4663, 2021 08 15.
Article de Anglais | MEDLINE | ID: mdl-34158358

RÉSUMÉ

PURPOSE: Mantle cell lymphoma (MCL) is a fatal subtype of non-Hodgkin lymphoma. SOX11 transcription factor is overexpressed in the majority of nodal MCL. We have previously reported that B cell-specific overexpression of SOX11 promotes MCL pathogenesis via critically increasing BCR signaling in vivo. SOX11 is an attractive target for MCL therapy; however, no small-molecule inhibitor of SOX11 has been identified to date. Although transcription factors are generally considered undruggable, the ability of SOX11 to bind to the minor groove of DNA led us to hypothesize that there may exist cavities at the protein-DNA interface that are amenable to targeting by small molecules. EXPERIMENTAL DESIGN: Using a combination of in silico predictions and experimental validations, we report here the discovery of three structurally related compounds (SOX11i) that bind SOX11, perturb its interaction with DNA, and effect SOX11-specific anti-MCL cytotoxicity. RESULTS: We find mechanistic validation of on-target activity of these SOX11i in the inhibition of BCR signaling and the transcriptional modulation of SOX11 target genes, specifically, in SOX11-expressing MCL cells. One of the three SOX11i exhibits relatively superior in vitro activity and displays cytotoxic synergy with ibrutinib in SOX11-expressing MCL cells. Importantly, this SOX11i induces cytotoxicity specifically in SOX11-positive ibrutinib-resistant MCL patient samples and inhibits Bruton tyrosine kinase phosphorylation in a xenograft mouse model derived from one of these subjects. CONCLUSIONS: Taken together, our results provide a foundation for therapeutically targeting SOX11 in MCL by a novel class of small molecules.


Sujet(s)
Lymphome à cellules du manteau/traitement médicamenteux , Facteurs de transcription SOX-C/antagonistes et inhibiteurs , Animaux , Humains , Souris , Cellules cancéreuses en culture
6.
Nat Struct Mol Biol ; 27(10): 913-924, 2020 10.
Article de Anglais | MEDLINE | ID: mdl-32807989

RÉSUMÉ

DNA polymerase ζ (Polζ) belongs to the same B-family as high-fidelity replicative polymerases, yet is specialized for the extension reaction in translesion DNA synthesis (TLS). Despite its importance in TLS, the structure of Polζ is unknown. We present cryo-EM structures of the Saccharomyces cerevisiae Polζ holoenzyme in the act of DNA synthesis (3.1 Å) and without DNA (4.1 Å). Polζ displays a pentameric ring-like architecture, with catalytic Rev3, accessory Pol31' Pol32 and two Rev7 subunits forming an uninterrupted daisy chain of protein-protein interactions. We also uncover the features that impose high fidelity during the nucleotide-incorporation step and those that accommodate mismatches and lesions during the extension reaction. Collectively, we decrypt the molecular underpinnings of Polζ's role in TLS and provide a framework for new cancer therapeutics.


Sujet(s)
Réparation de l'ADN/physiologie , Protéines de Saccharomyces cerevisiae/composition chimique , Domaine catalytique , Cryomicroscopie électronique , ADN/métabolisme , DNA polymerase III/composition chimique , DNA polymerase III/métabolisme , DNA-directed DNA polymerase/composition chimique , DNA-directed DNA polymerase/métabolisme , Modèles moléculaires , Conformation des protéines , Protéines de Saccharomyces cerevisiae/métabolisme
7.
Mol Cell ; 79(1): 180-190.e4, 2020 07 02.
Article de Anglais | MEDLINE | ID: mdl-32619468

RÉSUMÉ

Rigosertib is a styryl benzyl sulfone that inhibits growth of tumor cells and acts as a RAS mimetic by binding to Ras binding domains of RAS effectors. A recent study attributed rigosertib's mechanism of action to microtubule binding. In that study, rigosertib was obtained from a commercial vendor. We compared the purity of clinical-grade and commercially sourced rigosertib and found that commercially sourced rigosertib contains approximately 5% ON01500, a potent inhibitor of tubulin polymerization. Clinical-grade rigosertib, which is free of this impurity, does not exhibit tubulin-binding activity. Cell lines expressing mutant ß-tubulin have also been reported to be resistant to rigosertib. However, our study showed that these cells failed to proliferate in the presence of rigosertib at concentrations that are lethal to wild-type cells. Rigosertib induced a senescence-like phenotype in the small percentage of surviving cells, which could be incorrectly scored as resistant using short-term cultures.


Sujet(s)
Antinéoplasiques/pharmacologie , Prolifération cellulaire , Glycine/analogues et dérivés , Tumeurs du poumon/anatomopathologie , Sulfones/pharmacologie , Tubuline/métabolisme , Contamination de médicament , Résistance aux médicaments antinéoplasiques , Glycine/pharmacologie , Humains , Tumeurs du poumon/traitement médicamenteux , Tumeurs du poumon/métabolisme , Mutation , Tubuline/composition chimique , Tubuline/génétique , Cellules cancéreuses en culture
8.
Sci Adv ; 5(8): eaav0318, 2019 08.
Article de Anglais | MEDLINE | ID: mdl-31803841

RÉSUMÉ

JLP belongs to the JIP family whose members serve as scaffolding proteins that link motor proteins and their cargo for intracellular transport. Although JLP is mainly cytoplasmic, it accumulates as a focus in the perinuclear region when stimulated by extracellular stimuli. Focus formation, which changes the nucleus shape and concentrates the nuclear pores, depends on p38MAPK activation and the dynein retrograde motor protein complex. Extracellular stimuli trigger the tethering of PLK1 to the centrosome by JLP, leading to centrosome maturation and microtubule array formation. The centrosome localization domain of JLP is important for the binding of the centrosome and the formation of the JLP focus and the microtubule array. Furthermore, the formation of the JLP focus and the microtubule array is interdependent and important for the transport of NF-κB p65 to the nucleus and its unloading therein. In conclusion, JLP exhibits multiple functions in the nuclear translocation of NF-κB p65.


Sujet(s)
Protéines adaptatrices de la transduction du signal/métabolisme , Noyau de la cellule/métabolisme , Centrosome/métabolisme , Cytoplasme/métabolisme , Microtubules/métabolisme , Animaux , Cellules COS , Lignée cellulaire , Chlorocebus aethiops , Dynéines/métabolisme , Cellules HEK293 , Humains , Kinésine/métabolisme , Liaison aux protéines/physiologie , Transport des protéines/physiologie , Transduction du signal/physiologie , Facteur de transcription RelA
9.
Nat Struct Mol Biol ; 26(10): 955-962, 2019 10.
Article de Anglais | MEDLINE | ID: mdl-31582849

RÉSUMÉ

DNA polymerase δ (Polδ) plays pivotal roles in eukaryotic DNA replication and repair. Polδ is conserved from yeast to humans, and mutations in human Polδ have been implicated in various cancers. Saccharomyces cerevisiae Polδ consists of catalytic Pol3 and the regulatory Pol31 and Pol32 subunits. Here, we present the near atomic resolution (3.2 Å) cryo-EM structure of yeast Polδ holoenzyme in the act of DNA synthesis. The structure reveals an unexpected arrangement in which the regulatory subunits (Pol31 and Pol32) lie next to the exonuclease domain of Pol3 but do not engage the DNA. The Pol3 C-terminal domain contains a 4Fe-4S cluster and emerges as the keystone of Polδ assembly. We also show that the catalytic and regulatory subunits rotate relative to each other and that this is an intrinsic feature of the Polδ architecture. Collectively, the structure provides a framework for understanding DNA transactions at the replication fork.


Sujet(s)
DNA polymerase III/composition chimique , DNA-directed DNA polymerase/composition chimique , Protéines de Saccharomyces cerevisiae/composition chimique , Saccharomyces cerevisiae/composition chimique , Séquence d'acides aminés , Cryomicroscopie électronique , DNA polymerase III/métabolisme , DNA polymerase III/ultrastructure , ADN fongique/métabolisme , DNA-directed DNA polymerase/métabolisme , DNA-directed DNA polymerase/ultrastructure , Simulation de docking moléculaire , Liaison aux protéines , Conformation des protéines , Saccharomyces cerevisiae/métabolisme , Saccharomyces cerevisiae/ultrastructure , Protéines de Saccharomyces cerevisiae/métabolisme , Protéines de Saccharomyces cerevisiae/ultrastructure
10.
Curr Opin Struct Biol ; 53: 77-87, 2018 12.
Article de Anglais | MEDLINE | ID: mdl-30005324

RÉSUMÉ

The eukaryotic DNA replication machinery is conserved from yeast to humans and requires the actions of multiple DNA polymerases. In addition to replicative DNA polymerases for duplication of the leading and lagging DNA strands, another group of specialized polymerases is required for DNA repair and/or translesion DNA synthesis (TLS). We emphasize here recent findings that accelerate our understanding of the structure and mechanisms of these remarkable enzymes. We also highlight growing evidence on the role of DNA polymerases in the origin of certain cancers, and paradoxically as emerging targets for cancer therapy.


Sujet(s)
DNA-directed DNA polymerase , ADN/métabolisme , Cellules eucaryotes/enzymologie , Réparation de l'ADN , Réplication de l'ADN , DNA-directed DNA polymerase/composition chimique , DNA-directed DNA polymerase/métabolisme , DNA-directed DNA polymerase/physiologie , Domaines protéiques , Structure quaternaire des protéines
11.
Nat Med ; 24(6): 770-781, 2018 06.
Article de Anglais | MEDLINE | ID: mdl-29875463

RÉSUMÉ

Patients with metastatic cancer experience a severe loss of skeletal muscle mass and function known as cachexia. Cachexia is associated with poor prognosis and accelerated death in patients with cancer, yet its underlying mechanisms remain poorly understood. Here, we identify the metal-ion transporter ZRT- and IRT-like protein 14 (ZIP14) as a critical mediator of cancer-induced cachexia. ZIP14 is upregulated in cachectic muscles of mice and in patients with metastatic cancer and can be induced by TNF-α and TGF-ß cytokines. Strikingly, germline ablation or muscle-specific depletion of Zip14 markedly reduces muscle atrophy in metastatic cancer models. We find that ZIP14-mediated zinc uptake in muscle progenitor cells represses the expression of MyoD and Mef2c and blocks muscle-cell differentiation. Importantly, ZIP14-mediated zinc accumulation in differentiated muscle cells induces myosin heavy chain loss. These results highlight a previously unrecognized role for altered zinc homeostasis in metastatic cancer-induced muscle wasting and implicate ZIP14 as a therapeutic target for its treatment.


Sujet(s)
Cachexie/métabolisme , Cachexie/anatomopathologie , Transporteurs de cations/métabolisme , Muscles squelettiques/métabolisme , Muscles squelettiques/anatomopathologie , Tumeurs/métabolisme , Tumeurs/anatomopathologie , Régulation positive , Animaux , Différenciation cellulaire , Lignée cellulaire , Cytokines/métabolisme , Modèles animaux de maladie humaine , Humains , Souris de lignée C57BL , Chaînes lourdes de myosine/métabolisme , Métastase tumorale , Facteur de croissance transformant bêta/pharmacologie , Facteur de nécrose tumorale alpha/pharmacologie , Zinc/métabolisme
12.
Sci Rep ; 7(1): 1632, 2017 05 09.
Article de Anglais | MEDLINE | ID: mdl-28487506

RÉSUMÉ

The Zika virus (ZIKV) has emerged as a major health hazard. We present here a high resolution structure (1.55 Å) of ZIKV NS5 methyltransferase bound to a novel S-adenosylmethionine (SAM) analog in which a 4-fluorophenyl moiety substitutes for the methyl group. We show that the 4-fluorophenyl moiety extends into a portion of the RNA binding tunnel that typically contains the adenosine 2'OH of the RNA-cap moiety. Together, the new SAM analog and the high-resolution crystal structure are a step towards the development of antivirals against ZIKV and other flaviviruses.


Sujet(s)
Développement de médicament , Coiffes des ARN/métabolisme , Adémétionine/métabolisme , Virus Zika/enzymologie , Sites de fixation , Humains , Methyltransferases/composition chimique , Methyltransferases/métabolisme , Modèles moléculaires , Adémétionine/composition chimique , Thermodynamique , Protéines virales non structurales/métabolisme
13.
Sci Rep ; 7: 43904, 2017 03 08.
Article de Anglais | MEDLINE | ID: mdl-28272441

RÉSUMÉ

N1-methyl-deoxyadenosine (1-MeA) is formed by methylation of deoxyadenosine at the N1 atom. 1-MeA presents a block to replicative DNA polymerases due to its inability to participate in Watson-Crick (W-C) base pairing. Here we determine how human DNA polymerase-ι (Polι) promotes error-free replication across 1-MeA. Steady state kinetic analyses indicate that Polι is ~100 fold more efficient in incorporating the correct nucleotide T versus the incorrect nucleotide C opposite 1-MeA. To understand the basis of this selectivity, we determined ternary structures of Polι bound to template 1-MeA and incoming dTTP or dCTP. In both structures, template 1-MeA rotates to the syn conformation but pairs differently with dTTP versus dCTP. Thus, whereas dTTP partakes in stable Hoogsteen base pairing with 1-MeA, dCTP fails to gain a "foothold" and is largely disordered. Together, our kinetic and structural studies show how Polι maintains discrimination between correct and incorrect incoming nucleotide opposite 1-MeA in preserving genome integrity.


Sujet(s)
DNA-directed DNA polymerase/métabolisme , ADN/biosynthèse , Désoxyadénosine/métabolisme , Appariement de bases , Domaine catalytique , Cristallographie aux rayons X , ADN/composition chimique , DNA-directed DNA polymerase/composition chimique , Désoxyadénosine/composition chimique , Nucléotides désoxycytidyliques/composition chimique , Nucléotides désoxycytidyliques/métabolisme , Humains , Cinétique , Structure quaternaire des protéines , Nucléotides thymidyliques/composition chimique , Nucléotides thymidyliques/métabolisme ,
14.
Cell Rep ; 16(12): 3097-3102, 2016 09 20.
Article de Anglais | MEDLINE | ID: mdl-27633330

RÉSUMÉ

The Zika virus (ZIKV) poses a major public health emergency. To aid in the development of antivirals, we present two high-resolution crystal structures of the ZIKV NS5 methyltransferase: one bound to S-adenosylmethionine (SAM) and the other bound to SAM and 7-methyl guanosine diphosphate (7-MeGpp). We identify features of ZIKV NS5 methyltransferase that lend to structure-based antiviral drug discovery. Specifically, SAM analogs with functionalities on the Cß atom of the methionine portion of the molecules that occupy the RNA binding tunnel may provide better specificity relative to human RNA methyltransferases.


Sujet(s)
Methyltransferases/composition chimique , Protéines virales non structurales/composition chimique , Virus Zika/composition chimique , Virus Zika/enzymologie , Conformation des protéines
15.
Nat Struct Mol Biol ; 23(8): 752-4, 2016 08.
Article de Anglais | MEDLINE | ID: mdl-27399257

RÉSUMÉ

Zika virus has emerged as a pathogen of major health concern. Here, we present a high-resolution (1.62-Å) crystal structure of the RNA helicase from the French Polynesia strain. The structure is similar to that of the RNA helicase from Dengue virus, with variability in the conformations of loops typically involved in binding ATP and RNA. We identify druggable 'hotspots' that are well suited for in silico and/or fragment-based high-throughput drug discovery.


Sujet(s)
RNA helicases/composition chimique , Protéines virales/composition chimique , Virus Zika/enzymologie , Apoenzymes/composition chimique , Domaine catalytique , Cristallographie aux rayons X , Modèles moléculaires , Liaison aux protéines , Structure en hélice alpha , Similitude structurale de protéines
16.
Bioorg Med Chem ; 24(4): 521-44, 2016 Feb 15.
Article de Anglais | MEDLINE | ID: mdl-26762835

RÉSUMÉ

Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao, was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao.


Sujet(s)
Découverte de médicament , Indoles/pharmacologie , Inhibiteurs de protéines kinases/pharmacologie , Protein-Serine-Threonine Kinases/antagonistes et inhibiteurs , Pyrimidinones/pharmacologie , Relation dose-effet des médicaments , Humains , Indoles/synthèse chimique , Indoles/composition chimique , Structure moléculaire , Inhibiteurs de protéines kinases/synthèse chimique , Inhibiteurs de protéines kinases/composition chimique , Protein-Serine-Threonine Kinases/métabolisme , Pyrimidinones/synthèse chimique , Pyrimidinones/composition chimique , Relation structure-activité
17.
PLoS One ; 9(4): e94835, 2014.
Article de Anglais | MEDLINE | ID: mdl-24733111

RÉSUMÉ

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Here we report the ternary structure of the Polε catalytic subunit (Pol2) bound to a nascent G:C base pair (Pol2G:C). Pol2G:C has a typical B-family polymerase fold and embraces the template-primer duplex with the palm, fingers, thumb and exonuclease domains. The overall arrangement of domains is similar to the structure of Pol2T:A reported recently, but there are notable differences in their polymerase and exonuclease active sites. In particular, we observe Ca2+ ions at both positions A and B in the polymerase active site and also observe a Ca2+ at position B of the exonuclease site. We find that the contacts to the nascent G:C base pair in the Pol2G:C structure are maintained in the Pol2T:A structure and reflect the comparable fidelity of Pol2 for nascent purine-pyrimidine and pyrimidine-purine base pairs. We note that unlike that of Pol3, the shape of the nascent base pair binding pocket in Pol2 is modulated from the major grove side by the presence of Tyr431. Together with Pol2T:A, our results provide a framework for understanding the structural basis of high fidelity DNA synthesis by Pol2.


Sujet(s)
Domaine catalytique , DNA polymerase II/composition chimique , Saccharomyces cerevisiae/enzymologie , Cristallographie aux rayons X , DNA polymerase II/métabolisme , Nucléotides désoxycytidyliques/composition chimique , Nucléotides désoxycytidyliques/métabolisme , Similitude structurale de protéines
18.
J Mol Biol ; 426(2): 301-8, 2014 Jan 23.
Article de Anglais | MEDLINE | ID: mdl-24144619

RÉSUMÉ

DNA polymerase ε (Polε) is a multi-subunit polymerase that contributes to genomic stability via its roles in leading strand replication and the repair of damaged DNA. Polε from Saccharomyces cerevisiae is composed of four subunits--Pol2, Dpb2, Dpb3, and Dpb4. Here, we report the presence of a [Fe-S] cluster directly within the active polymerase domain of Pol2 (residues 1-1187). We show that binding of the [Fe-S] cluster is mediated by cysteines in an insertion (Pol2(ins)) that is conserved in Pol2 orthologs but is absent in the polymerase domains of Polα, Polδ, and Polζ. We also show that the [Fe-S] cluster is required for Pol2 polymerase activity but not for its exonuclease activity. Collectively, our work suggests that Polε is perhaps more sensitive than other DNA polymerases to changes in oxidative stress in eukaryotic cells.


Sujet(s)
DNA polymerase II/composition chimique , DNA polymerase II/métabolisme , Ferrosulfoprotéines/composition chimique , Ferrosulfoprotéines/métabolisme , Fer/analyse , Saccharomyces cerevisiae/enzymologie , Soufre/analyse , Stress oxydatif
19.
Cell Rep ; 5(1): 79-86, 2013 Oct 17.
Article de Anglais | MEDLINE | ID: mdl-24120860

RÉSUMÉ

DNA polymerase ζ (Polζ) is specialized for the extension step of translesion DNA synthesis (TLS). Despite its central role in maintaining genome integrity, little is known about its overall architecture. Initially identified as a heterodimer of the catalytic subunit Rev3 and the accessory subunit Rev7, yeast Polζ has recently been shown to form a stable four-subunit enzyme (Polζ-d) upon the incorporation of Pol31 and Pol32, the accessory subunits of yeast Polδ. To understand the 3D architecture and assembly of Polζ and Polζ-d, we employed electron microscopy. We show here how the catalytic and accessory subunits of Polζ and Polζ-d are organized relative to each other. In particular, we show that Polζ-d has a bilobal architecture resembling the replicative polymerases and that Pol32 lies in proximity to Rev7. Collectively, our study provides views of Polζ and Polζ-d and a structural framework for understanding their roles in DNA damage bypass.


Sujet(s)
Protéines fongiques/métabolisme , Levures/enzymologie , Cristallographie aux rayons X , ADN/composition chimique , ADN/génétique , ADN/métabolisme , Altération de l'ADN , Réplication de l'ADN , DNA-directed DNA polymerase/composition chimique , DNA-directed DNA polymerase/génétique , DNA-directed DNA polymerase/isolement et purification , DNA-directed DNA polymerase/métabolisme , Protéines fongiques/composition chimique , Protéines fongiques/génétique , Humains , Modèles moléculaires , Liaison aux protéines , Structure secondaire des protéines , Saccharomyces cerevisiae/composition chimique , Saccharomyces cerevisiae/enzymologie , Saccharomyces cerevisiae/génétique , Protéines de Saccharomyces cerevisiae/composition chimique , Protéines de Saccharomyces cerevisiae/génétique , Protéines de Saccharomyces cerevisiae/métabolisme , Levures/composition chimique , Levures/génétique
20.
Nat Struct Mol Biol ; 19(6): 628-32, 2012 May 06.
Article de Anglais | MEDLINE | ID: mdl-22562137

RÉSUMÉ

A major clinical problem in the use of cisplatin to treat cancers is tumor resistance. DNA polymerase η (Pol-η) is a crucial polymerase that allows cancer cells to cope with the cisplatin-DNA adducts that are formed during chemotherapy. We present here a structure of human Pol-η inserting deoxycytidine triphosphate (dCTP) opposite a cisplatin intrastrand cross-link (PtGpG). We show that the specificity of human Pol-η for PtGpG derives from an active site that is open to permit Watson-Crick geometry of the nascent PtGpG-dCTP base pair and to accommodate the lesion without steric hindrance. This specificity is augmented by the residues Gln38 and Ser62, which interact with PtGpG, and Arg61, which interacts with the incoming dCTP. Collectively, the structure provides a basis for understanding how Pol-η in human cells can tolerate the DNA damage caused by cisplatin chemotherapy and offers a framework for the design of inhibitors in cancer therapy.


Sujet(s)
Antinéoplasiques/pharmacologie , Cisplatine/pharmacologie , Adduits à l'ADN/synthèse chimique , DNA-directed DNA polymerase/composition chimique , DNA-directed DNA polymerase/métabolisme , Nucléotides désoxycytidyliques/métabolisme , Domaine catalytique , Cisplatine/synthèse chimique , Cisplatine/composition chimique , Cisplatine/métabolisme , Cristallographie aux rayons X , Adduits à l'ADN/composition chimique , Adduits à l'ADN/métabolisme , Nucléotides désoxycytidyliques/composition chimique , Humains , Modèles moléculaires , Tumeurs/traitement médicamenteux , Spécificité du substrat
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