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2.
Nat Commun ; 12(1): 4671, 2021 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-34344863

RESUMO

Triple negative breast cancer (TNBC) remains challenging because of heterogeneous responses to chemotherapy. Incomplete response is associated with a greater risk of metastatic progression. Therefore, treatments that target chemotherapy-resistant TNBC and enhance chemosensitivity would improve outcomes for these high-risk patients. Breast cancer stem cell-like cells (BCSCs) have been proposed to represent a chemotherapy-resistant subpopulation responsible for tumor initiation, progression and metastases. Targeting this population could lead to improved TNBC disease control. Here, we describe a novel multi-kinase inhibitor, 108600, that targets the TNBC BCSC population. 108600 treatment suppresses growth, colony and mammosphere forming capacity of BCSCs and induces G2M arrest and apoptosis of TNBC cells. In vivo, 108600 treatment of mice bearing triple negative tumors results in the induction of apoptosis and overcomes chemotherapy resistance. Finally, treatment with 108600 and chemotherapy suppresses growth of pre-established TNBC metastases, providing additional support for the clinical translation of this agent to clinical trials.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Células-Tronco Neoplásicas/efeitos dos fármacos , Nitrobenzenos/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Tiazinas/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Caseína Quinase II/antagonistas & inibidores , Caseína Quinase II/química , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Camundongos , Células-Tronco Neoplásicas/patologia , Nitrobenzenos/química , Nitrobenzenos/farmacologia , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/química , Tiazinas/química , Tiazinas/farmacologia , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Dev Cell ; 56(15): 2207-2222.e7, 2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34256011

RESUMO

Cells counter DNA damage through repair or apoptosis, yet a direct mechanism for this choice has remained elusive. When facing interstrand crosslinks (ICLs), the ICL-repair protein FANCI heterodimerizes with FANCD2 to initiate ICL excision. We found that FANCI alternatively interacts with a pro-apoptotic factor, PIDD1, to enable PIDDosome (PIDD1-RAIDD-caspase-2) formation and apoptotic death. FANCI switches from FANCD2/repair to PIDD1/apoptosis signaling in the event of ICL-repair failure. Specifically, removing key endonucleases downstream of FANCI/FANCD2, increasing ICL levels, or allowing damaged cells into mitosis (when repair is suppressed) all suffice for switching. Reciprocally, apoptosis-committed FANCI reverts from PIDD1 to FANCD2 after a failed attempt to assemble the PIDDosome. Monoubiquitination and deubiquitination at FANCI K523 impact interactor selection. These data unveil a repair-or-apoptosis switch in eukaryotes. Beyond ensuring the removal of unrepaired genomes, the switch's bidirectionality reveals that damaged cells can offset apoptotic defects via de novo attempts at lesion repair.

4.
Nature ; 595(7867): 444-449, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34194047

RESUMO

The size of the transcriptional program of long non-coding RNAs in the mammalian genome has engendered discussions about their biological roles1, particularly the promoter antisense (PAS) transcripts2,3. Here we report the development of an assay-referred to as chromatin isolation by RNA-Cas13a complex-to quantitatively detect the distribution of RNA in the genome. The assay revealed that PAS RNAs serve as a key gatekeeper of a broad transcriptional pause release program, based on decommissioning the 7SK small nuclear RNA-dependent inhibitory P-TEFb complex. Induction of PAS RNAs by liganded ERα led to a significant loss of H3K9me3 and the release of basally recruited HP1α and KAP1 on activated target gene promoters. This release was due to PAS RNA-dependent recruitment of H3K9me3 demethylases, which required interactions with a compact stem-loop structure in the PAS RNAs, an apparent feature of similarly regulated PAS RNAs. Activation of the ERα-bound MegaTrans enhancer, which is essential for robust pause release, required the recruitment of phosphorylated KAP1, with its transfer to the cognate promoters permitting 17ß-oestradiol-induced pause release and activation of the target gene. This study reveals a mechanism, based on RNA structure, that mediates the function of PAS RNAs in gene regulation.

5.
Nat Commun ; 12(1): 4020, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34188055

RESUMO

PrimPol is a human DNA polymerase-primase that localizes to mitochondria and nucleus and bypasses the major oxidative lesion 7,8-dihydro-8-oxoguanine (oxoG) via translesion synthesis, in mostly error-free manner. We present structures of PrimPol insertion complexes with a DNA template-primer and correct dCTP or erroneous dATP opposite the lesion, as well as extension complexes with C or A as a 3'-terminal primer base. We show that during the insertion of C and extension from it, the active site is unperturbed, reflecting the readiness of PrimPol to accommodate oxoG(anti). The misinsertion of A opposite oxoG(syn) also does not alter the active site, and is likely less favorable due to lower thermodynamic stability of the oxoG(syn)•A base-pair. During the extension step, oxoG(syn) induces an opening of its base-pair with A or misalignment of the 3'-A primer terminus. Together, the structures show how PrimPol accurately synthesizes DNA opposite oxidatively damaged DNA in human cells.


Assuntos
Pareamento de Bases/genética , Dano ao DNA/genética , DNA Primase/metabolismo , Replicação do DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , Guanina/análogos & derivados , Enzimas Multifuncionais/metabolismo , Guanina/química , Humanos , Mitocôndrias/enzimologia , Mitocôndrias/genética , Estresse Oxidativo/genética , Conformação Proteica , Espécies Reativas de Oxigênio/metabolismo
6.
Clin Cancer Res ; 27(16): 4652-4663, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34158358

RESUMO

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.

7.
Nat Struct Mol Biol ; 27(10): 913-924, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32807989

RESUMO

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.


Assuntos
Reparo do DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/química , Domínio Catalítico , Microscopia Crioeletrônica , DNA/metabolismo , DNA Polimerase III/química , DNA Polimerase III/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
Mol Cell ; 79(1): 180-190.e4, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32619468

RESUMO

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.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células , Glicina/análogos & derivados , Neoplasias Pulmonares/patologia , Sulfonas/farmacologia , Tubulina (Proteína)/metabolismo , Contaminação de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Glicina/farmacologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Mutação , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Células Tumorais Cultivadas
9.
Nat Microbiol ; 5(1): 166-180, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31768029

RESUMO

Clostridioides (formerly Clostridium) difficile is a leading cause of healthcare-associated infections. Although considerable progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we perform a comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observe a high level of epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity, the corresponding gene of which is highly conserved across our dataset and in all of the approximately 300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacts sporulation, a key step in C. difficile disease transmission, and these results are consistently supported by multiomics data, genetic experiments and a mouse colonization model. Further experimental and transcriptomic analyses suggest that epigenetic regulation is associated with cell length, biofilm formation and host colonization. These findings provide a unique epigenetic dimension to characterize medically relevant biological processes in this important pathogen. This study also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomic studies.


Assuntos
Clostridioides difficile/enzimologia , Clostridioides difficile/fisiologia , Clostridioides difficile/patogenicidade , Metilases de Modificação do DNA/metabolismo , Epigênese Genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridioides difficile/genética , Infecções por Clostridium/microbiologia , Cricetinae , Metilação de DNA , Metilases de Modificação do DNA/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Epigenoma , Regulação Bacteriana da Expressão Gênica , Variação Genética , Genoma Bacteriano/genética , Humanos , Camundongos , Mutação , Motivos de Nucleotídeos , Filogenia , Elementos Reguladores de Transcrição/genética , Esporos Bacterianos/genética , Esporos Bacterianos/fisiologia , Especificidade por Substrato
10.
Sci Rep ; 9(1): 16400, 2019 11 08.
Artigo em Inglês | MEDLINE | ID: mdl-31704958

RESUMO

Cytarabine (AraC) is the mainstay chemotherapy for acute myeloid leukemia (AML). Whereas initial treatment with AraC is usually successful, most AML patients tend to relapse, and AraC treatment-induced mutagenesis may contribute to the development of chemo-resistant leukemic clones. We show here that whereas the high-fidelity replicative polymerase Polδ is blocked in the replication of AraC, the lower-fidelity translesion DNA synthesis (TLS) polymerase Polη is proficient, inserting both correct and incorrect nucleotides opposite a template AraC base. Furthermore, we present high-resolution crystal structures of human Polη with a template AraC residue positioned opposite correct (G) and incorrect (A) incoming deoxynucleotides. We show that Polη can accommodate local perturbation caused by the AraC via specific hydrogen bonding and maintain a reaction-ready active site alignment for insertion of both correct and incorrect incoming nucleotides. Taken together, the structures provide a novel basis for the ability of Polη to promote AraC induced mutagenesis in relapsed AML patients.


Assuntos
Citarabina/farmacologia , DNA Polimerase II/química , DNA Polimerase II/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Citarabina/análogos & derivados , Citarabina/química , Replicação do DNA/efeitos dos fármacos , Humanos , Modelos Moleculares , Estrutura Molecular , Conformação Proteica
11.
Nat Struct Mol Biol ; 26(10): 955-962, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31582849

RESUMO

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.


Assuntos
DNA Polimerase III/química , DNA Polimerase Dirigida por DNA/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Sequência de Aminoácidos , Microscopia Crioeletrônica , DNA Polimerase III/metabolismo , DNA Polimerase III/ultraestrutura , DNA Fúngico/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/ultraestrutura , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura
12.
Nat Struct Mol Biol ; 26(3): 193-203, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30833784

RESUMO

A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of enhancer assembly in signaling events remain poorly understood. Here we report that in human breast cancer cells, the acute 17ß-estradiol-dependent activation of functional enhancers requires assembly of an enhancer RNA-dependent ribonucleoprotein (eRNP) complex exhibiting properties of phase-separated condensates. Unexpectedly, while acute ligand-dependent assembly of eRNPs resulted in enhancer activation sensitive to chemical disruption of phase separation, chronically activated enhancers proved resistant to such disruption, with progressive maturation of eRNPs to a more gel-like state. Acute, but not chronic, stimulation resulted in ligand-induced, condensin-dependent changes in spatial chromatin conformation based on homotypic enhancer association, resulting in cooperative enhancer-activation events. Thus, distinct physicochemical properties of eRNP condensates on enhancers serve as determinants of rapid ligand-dependent alterations in chromosomal architecture and cooperative enhancer activation.


Assuntos
Elementos Facilitadores Genéticos/genética , Estradiol/metabolismo , Ribonucleoproteínas/metabolismo , Ativação Transcricional/fisiologia , Linhagem Celular Tumoral , Cromatina , Cromossomos/fisiologia , Humanos , Células MCF-7 , Conformação Proteica , Transcrição Genética/genética , Ativação Transcricional/genética
13.
Sci Rep ; 8(1): 12702, 2018 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-30140014

RESUMO

Cytarabine (AraC) is an essential chemotherapeutic for acute myeloid leukemia (AML) and resistance to this drug is a major cause of treatment failure. AraC is a nucleoside analog that differs from 2'-deoxycytidine only by the presence of an additional hydroxyl group at the C2' position of the 2'-deoxyribose. The active form of the drug AraC 5'-triphosphate (AraCTP) is utilized by human replicative DNA polymerases to insert AraC at the 3' terminus of a growing DNA chain. This impedes further primer extension and is a primary basis for the drug action. The Y-family translesion synthesis (TLS) DNA polymerase η (Polη) counteracts this barrier to DNA replication by efficient extension from AraC-terminated primers. Here, we provide high-resolution structures of human Polη with AraC incorporated at the 3'-primer terminus. We show that Polη can accommodate AraC at different stages of the catalytic cycle, and that it can manipulate the conformation of the AraC sugar via specific hydrogen bonding and stacking interactions. Taken together, the structures provide a basis for the ability of Polη to extend DNA synthesis from AraC terminated primers.


Assuntos
Antineoplásicos/química , Citarabina/química , DNA Polimerase Dirigida por DNA/metabolismo , Antineoplásicos/farmacologia , Cristalização , Citarabina/farmacologia , Citidina/química , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Replicação do DNA/genética , Desoxicitidina/química , Humanos , Estrutura Molecular , Difração de Raios X
14.
Mol Cell ; 71(4): 526-539.e8, 2018 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-30118678

RESUMO

Nuclear receptors induce both transcriptional activation and repression programs responsible for development, homeostasis, and disease. Here, we report a previously overlooked enhancer decommissioning strategy underlying a large estrogen receptor alpha (ERα)-dependent transcriptional repression program. The unexpected signature for this E2-induced program resides in indirect recruitment of ERα to a large cohort of pioneer factor basally active FOXA1-bound enhancers that lack cognate ERα DNA-binding elements. Surprisingly, these basally active estrogen-repressed (BAER) enhancers are decommissioned by ERα-dependent recruitment of the histone demethylase KDM2A, functioning independently of its demethylase activity. Rather, KDM2A tethers the E3 ubiquitin-protein ligase NEDD4 to ubiquitylate/dismiss Pol II to abrogate eRNA transcription, with consequent target gene downregulation. Thus, our data reveal that Pol II ubiquitylation/dismissal may serve as a potentially broad strategy utilized by indirectly bound nuclear receptors to abrogate large programs of pioneer factor-mediated, eRNA-producing enhancers.


Assuntos
Elementos Facilitadores Genéticos , Receptor alfa de Estrogênio/genética , Proteínas F-Box/genética , Fator 3-alfa Nuclear de Hepatócito/genética , Histona Desmetilases com o Domínio Jumonji/genética , Ubiquitina-Proteína Ligases Nedd4/genética , RNA Polimerase II/genética , Sequência de Bases , Sítios de Ligação , Sistemas CRISPR-Cas , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Proteínas F-Box/metabolismo , Edição de Genes/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji/metabolismo , Células MCF-7 , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Ligação Proteica , RNA/genética , RNA/metabolismo , RNA Polimerase II/metabolismo , Transdução de Sinais , Transcrição Genética/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacos
15.
Curr Opin Struct Biol ; 53: 77-87, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30005324

RESUMO

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.


Assuntos
DNA Polimerase Dirigida por DNA , DNA/metabolismo , Células Eucarióticas/enzimologia , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Domínios Proteicos , Estrutura Quaternária de Proteína
16.
Nat Commun ; 8(1): 965, 2017 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-29042535

RESUMO

Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke which, after metabolic activation, can react with the exocyclic N 2 amino group of guanine to generate four stereoisomeric BP-N 2-dG adducts. Rev1 is unique among translesion synthesis DNA polymerases in employing a protein-template-directed mechanism of DNA synthesis opposite undamaged and damaged guanine. Here we report high-resolution structures of yeast Rev1 with three BP-N 2-dG adducts, namely the 10S (+)-trans-BP-N 2-dG, 10R (+)-cis-BP-N 2-dG, and 10S ( - )-cis-BP-N 2-dG. Surprisingly, in all three structures, the bulky and hydrophobic BP pyrenyl residue is entirely solvent-exposed in the major groove of the DNA. This is very different from the adduct alignments hitherto observed in free or protein-bound DNA. All complexes are well poised for dCTP insertion. Our structures provide a view of cis-BP-N 2-dG adducts in a DNA polymerase active site, and offer a basis for understanding error-free replication of the BP-derived stereoisomeric guanine adducts.Benzo[a]pyrene (BP) is a carcinogen in cigarette smoke that upon metabolic activation reacts with guanine. Here, the authors present the structures of the translesion DNA synthesis polymerase Rev1 in complex with three of the four possible stereoisomeric BP-N 2 -dG adducts, which gives insights how Rev1 achieves error-free replication.


Assuntos
Benzo(a)pireno/química , Benzo(a)pireno/metabolismo , Nucleotidiltransferases/química , Nucleotidiltransferases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Domínio Catalítico , Cristalografia por Raios X , Adutos de DNA/química , Reparo do DNA/fisiologia , Replicação do DNA , Guanina/química , Metais/química , Metais/metabolismo , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Conformação Proteica , Estereoisomerismo
17.
J Am Chem Soc ; 139(35): 12219-12227, 2017 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-28780862

RESUMO

Many intrinsically disordered proteins (IDPs) and protein regions (IDRs) engage in transient, yet specific, interactions with a variety of protein partners. Often, if not always, interactions with a protein partner lead to partial folding of the IDR. Characterizing the conformational space of such complexes is challenging: in solution-state NMR, signals of the IDR in the interacting region become broad, weak, and often invisible, while X-ray crystallography only provides information on fully ordered regions. There is thus a need for a simple method to characterize both fully and partially ordered regions in the bound state of IDPs. Here, we introduce an approach based on monitoring chemical exchange by NMR to investigate the state of an IDR that folds upon binding through the observation of the free state of the protein. Structural constraints for the bound state are obtained from chemical shifts, and site-specific dynamics of the bound state are characterized by relaxation rates. The conformation of the interacting part of the IDR was determined and subsequently docked onto the structure of the folded partner. We apply the method to investigate the interaction between the disordered C-terminal region of Artemis and the DNA binding domain of Ligase IV. We show that we can accurately reproduce the structure of the core of the complex determined by X-ray crystallography and identify a broader interface. The method is widely applicable to the biophysical investigation of complexes of disordered proteins and folded proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Ressonância Magnética Nuclear Biomolecular/métodos , Cristalografia por Raios X , DNA Ligase Dependente de ATP/química , Simulação de Acoplamento Molecular , Ligação Proteica , Conformação Proteica , Dobramento de Proteína
18.
Nat Rev Mol Cell Biol ; 18(8): 471-476, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28537575

RESUMO

The idea that signal-dependent transcription might involve the generation of transient DNA nicks or even breaks in the regulatory regions of genes, accompanied by activation of DNA damage repair pathways, would seem to be counterintuitive, as DNA damage is usually considered harmful to cellular integrity. However, recent studies have generated a substantial body of evidence that now argues that programmed DNA single- or double-strand breaks can, at least in specific cases, have a role in transcription regulation. Here, we discuss the emerging functions of DNA breaks in the relief of DNA torsional stress and in promoter and enhancer activation.


Assuntos
Dano ao DNA/genética , DNA/química , Animais , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Regulação da Expressão Gênica , Humanos
19.
Sci Rep ; 7(1): 1632, 2017 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-28487506

RESUMO

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.


Assuntos
Desenvolvimento de Medicamentos , Capuzes de RNA/metabolismo , S-Adenosilmetionina/metabolismo , Zika virus/enzimologia , Sítios de Ligação , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , S-Adenosilmetionina/química , Termodinâmica , Proteínas não Estruturais Virais/metabolismo
20.
Sci Rep ; 7: 43904, 2017 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-28272441

RESUMO

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.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Desoxiadenosinas/metabolismo , Pareamento de Bases , Domínio Catalítico , Cristalografia por Raios X , DNA/química , DNA Polimerase Dirigida por DNA/química , Desoxiadenosinas/química , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxicitosina/metabolismo , Humanos , Cinética , Estrutura Quaternária de Proteína , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo
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