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1.
Nature ; 604(7907): 749-756, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35444283

RESUMO

Amplification of the CCNE1 locus on chromosome 19q12 is prevalent in multiple tumour types, particularly in high-grade serous ovarian cancer, uterine tumours and gastro-oesophageal cancers, where high cyclin E levels are associated with genome instability, whole-genome doubling and resistance to cytotoxic and targeted therapies1-4. To uncover therapeutic targets for tumours with CCNE1 amplification, we undertook genome-scale CRISPR-Cas9-based synthetic lethality screens in cellular models of CCNE1 amplification. Here we report that increasing CCNE1 dosage engenders a vulnerability to the inhibition of the PKMYT1 kinase, a negative regulator of CDK1. To inhibit PKMYT1, we developed RP-6306, an orally bioavailable and selective inhibitor that shows single-agent activity and durable tumour regressions when combined with gemcitabine in models of CCNE1 amplification. RP-6306 treatment causes unscheduled activation of CDK1 selectively in CCNE1-overexpressing cells, promoting early mitosis in cells undergoing DNA synthesis. CCNE1 overexpression disrupts CDK1 homeostasis at least in part through an early activation of the MMB-FOXM1 mitotic transcriptional program. We conclude that PKMYT1 inhibition is a promising therapeutic strategy for CCNE1-amplified cancers.


Assuntos
Ciclina E , Proteínas de Membrana , Neoplasias Ovarianas , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases , Proteína Quinase CDC2 , Ciclina E/genética , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Membrana/genética , Neoplasias/genética , Neoplasias Ovarianas/patologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Mutações Sintéticas Letais
2.
Nat Cancer ; 2(12): 1357-1371, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-35121901

RESUMO

BRCA1/2-mutated cancer cells adapt to the genome instability caused by their deficiency in homologous recombination (HR). Identification of these adaptive mechanisms may provide therapeutic strategies to target tumors caused by the loss of these genes. In the present study, we report genome-scale CRISPR-Cas9 synthetic lethality screens in isogenic pairs of BRCA1- and BRCA2-deficient cells and identify CIP2A as an essential gene in BRCA1- and BRCA2-mutated cells. CIP2A is cytoplasmic in interphase but, in mitosis, accumulates at DNA lesions as part of a complex with TOPBP1, a multifunctional genome stability factor. Unlike PARP inhibition, CIP2A deficiency does not cause accumulation of replication-associated DNA lesions that require HR for their repair. In BRCA-deficient cells, the CIP2A-TOPBP1 complex prevents lethal mis-segregation of acentric chromosomes that arises from impaired DNA synthesis. Finally, physical disruption of the CIP2A-TOPBP1 complex is highly deleterious in BRCA-deficient tumors, indicating that CIP2A represents an attractive synthetic lethal therapeutic target for BRCA1- and BRCA2-mutated cancers.


Assuntos
Neoplasias , Mutações Sintéticas Letais , Proteínas de Transporte/genética , Instabilidade Cromossômica , DNA , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica/genética , Recombinação Homóloga , Humanos , Proteínas Nucleares/genética
3.
Nat Commun ; 11(1): 6233, 2020 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-33277478

RESUMO

The KEOPS complex, which is conserved across archaea and eukaryotes, is composed of four core subunits; Pcc1, Kae1, Bud32 and Cgi121. KEOPS is crucial for the fitness of all organisms examined. In humans, pathogenic mutations in KEOPS genes lead to Galloway-Mowat syndrome, an autosomal-recessive disease causing childhood lethality. Kae1 catalyzes the universal and essential tRNA modification N6-threonylcarbamoyl adenosine, but the precise roles of all other KEOPS subunits remain an enigma. Here we show using structure-guided studies that Cgi121 recruits tRNA to KEOPS by binding to its 3' CCA tail. A composite model of KEOPS bound to tRNA reveals that all KEOPS subunits form an extended tRNA-binding surface that we have validated in vitro and in vivo to mediate the interaction with the tRNA substrate and its modification. These findings provide a framework for understanding the inner workings of KEOPS and delineate why all KEOPS subunits are essential.


Assuntos
Proteínas Arqueais/química , Methanocaldococcus/metabolismo , Complexos Multiproteicos/química , RNA de Transferência/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Cristalografia por Raios X , Methanocaldococcus/genética , Modelos Moleculares , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA de Transferência de Lisina/química , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/metabolismo
4.
Cell ; 182(2): 481-496.e21, 2020 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-32649862

RESUMO

The response to DNA damage is critical for cellular homeostasis, tumor suppression, immunity, and gametogenesis. In order to provide an unbiased and global view of the DNA damage response in human cells, we undertook 31 CRISPR-Cas9 screens against 27 genotoxic agents in the retinal pigment epithelium-1 (RPE1) cell line. These screens identified 890 genes whose loss causes either sensitivity or resistance to DNA-damaging agents. Mining this dataset, we discovered that ERCC6L2 (which is mutated in a bone-marrow failure syndrome) codes for a canonical non-homologous end-joining pathway factor, that the RNA polymerase II component ELOF1 modulates the response to transcription-blocking agents, and that the cytotoxicity of the G-quadruplex ligand pyridostatin involves trapping topoisomerase II on DNA. This map of the DNA damage response provides a rich resource to study this fundamental cellular system and has implications for the development and use of genotoxic agents in cancer therapy.


Assuntos
Dano ao DNA , Redes Reguladoras de Genes/fisiologia , Aminoquinolinas/farmacologia , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Citocromo-B(5) Redutase/genética , Citocromo-B(5) Redutase/metabolismo , Dano ao DNA/efeitos dos fármacos , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , DNA Topoisomerases Tipo II/genética , DNA Topoisomerases Tipo II/metabolismo , Humanos , Camundongos , Ácidos Picolínicos/farmacologia , RNA Guia de Cinetoplastídeos/metabolismo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética
5.
Nature ; 560(7716): 117-121, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30022168

RESUMO

53BP1 is a chromatin-binding protein that regulates the repair of DNA double-strand breaks by suppressing the nucleolytic resection of DNA termini1,2. This function of 53BP1 requires interactions with PTIP3 and RIF14-9, the latter of which recruits REV7 (also known as MAD2L2) to break sites10,11. How 53BP1-pathway proteins shield DNA ends is currently unknown, but there are two models that provide the best potential explanation of their action. In one model the 53BP1 complex strengthens the nucleosomal barrier to end-resection nucleases12,13, and in the other 53BP1 recruits effector proteins with end-protection activity. Here we identify a 53BP1 effector complex, shieldin, that includes C20orf196 (also known as SHLD1), FAM35A (SHLD2), CTC-534A2.2 (SHLD3) and REV7. Shieldin localizes to double-strand-break sites in a 53BP1- and RIF1-dependent manner, and its SHLD2 subunit binds to single-stranded DNA via OB-fold domains that are analogous to those of RPA1 and POT1. Loss of shieldin impairs non-homologous end-joining, leads to defective immunoglobulin class switching and causes hyper-resection. Mutations in genes that encode shieldin subunits also cause resistance to poly(ADP-ribose) polymerase inhibition in BRCA1-deficient cells and tumours, owing to restoration of homologous recombination. Finally, we show that binding of single-stranded DNA by SHLD2 is critical for shieldin function, consistent with a model in which shieldin protects DNA ends to mediate 53BP1-dependent DNA repair.


Assuntos
Reparo do DNA , Complexos Multiproteicos/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem Celular , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples/genética , Feminino , Genes BRCA1 , Humanos , Switching de Imunoglobulina/genética , Camundongos , Modelos Biológicos , Complexos Multiproteicos/química , Complexos Multiproteicos/deficiência , Complexos Multiproteicos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Proteínas de Ligação a Telômeros/metabolismo , Proteína Supressora de Tumor p53/deficiência
6.
Elife ; 62017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28826474

RESUMO

DNA double-strand breaks (DSBs) and short telomeres are structurally similar, yet they have diametrically opposed fates. Cells must repair DSBs while blocking the action of telomerase on these ends. Short telomeres must avoid recognition by the DNA damage response while promoting telomerase recruitment. In Saccharomyces cerevisiae, the Pif1 helicase, a telomerase inhibitor, lies at the interface of these end-fate decisions. Using Pif1 as a sensor, we uncover a transition point in which 34 bp of telomeric (TG1-3)n repeat sequence renders a DNA end insensitive to Pif1 action, thereby enabling extension by telomerase. A similar transition point exists at natural chromosome ends, where telomeres shorter than ~40 bp are inefficiently extended by telomerase. This phenomenon is not due to known Pif1 modifications and we instead propose that Cdc13 renders TG34+ ends insensitive to Pif1 action. We contend that the observed threshold of Pif1 activity defines a dividing line between DSBs and telomeres.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Telômero/metabolismo , Sequência de Bases , Cromossomos , DNA Fúngico , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos/genética , Vetores Genéticos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutagênese , Mutação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Telomerase/metabolismo , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
7.
Nucleic Acids Res ; 45(2): 805-817, 2017 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-27903914

RESUMO

The KEOPS/EKC complex is a tRNA modification complex involved in the biosynthesis of N6-threonylcarbamoyladenosine (t6A), a universally conserved tRNA modification found on ANN-codon recognizing tRNAs. In archaea and eukaryotes, KEOPS is composed of OSGEP/Kae1, PRPK/Bud32, TPRKB/Cgi121 and LAGE3/Pcc1. In fungi, KEOPS contains an additional subunit, Gon7, whose orthologs outside of fungi, if existent, remain unidentified. In addition to displaying defective t6A biosynthesis, Saccharomyces cerevisiae strains harboring KEOPS mutations are compromised for telomere homeostasis, growth and transcriptional co-activation. To identify a Gon7 ortholog in multicellular eukaryotes as well as to uncover KEOPS-interacting proteins that may link t6A biosynthesis to the diverse set of KEOPS mutant phenotypes, we conducted a proteomic analysis of human KEOPS. This work identified 152 protein interactors, one of which, C14ORF142, interacted strongly with all four KEOPS subunits, suggesting that it may be a core component of human KEOPS. Further characterization of C14ORF142 revealed that it shared a number of biophysical and biochemical features with fungal Gon7, suggesting that C14ORF142 is the human ortholog of Gon7. In addition, our proteomic analysis identified specific interactors for different KEOPS subcomplexes, hinting that individual KEOPS subunits may have additional functions outside of t6A biosynthesis.


Assuntos
Complexos Multiproteicos , Fases de Leitura Aberta , Subunidades Proteicas , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Humanos , Proteínas Intrinsicamente Desordenadas/metabolismo , Complexos Multiproteicos/química , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/química
8.
Nucleic Acids Res ; 41(12): 6332-46, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23620299

RESUMO

The universally conserved Kae1/Qri7/YgjD and Sua5/YrdC protein families have been implicated in growth, telomere homeostasis, transcription and the N6-threonylcarbamoylation (t(6)A) of tRNA, an essential modification required for translational fidelity by the ribosome. In bacteria, YgjD orthologues operate in concert with the bacterial-specific proteins YeaZ and YjeE, whereas in archaeal and eukaryotic systems, Kae1 operates as part of a larger macromolecular assembly called KEOPS with Bud32, Cgi121, Gon7 and Pcc1 subunits. Qri7 orthologues function in the mitochondria and may represent the most primitive member of the Kae1/Qri7/YgjD protein family. In accordance with previous findings, we confirm that Qri7 complements Kae1 function and uncover that Qri7 complements the function of all KEOPS subunits in growth, t(6)A biosynthesis and, to a partial degree, telomere maintenance. These observations suggest that Kae1 provides a core essential function that other subunits within KEOPS have evolved to support. Consistent with this inference, Qri7 alone is sufficient for t(6)A biosynthesis with Sua5 in vitro. In addition, the 2.9 Å crystal structure of Qri7 reveals a simple homodimer arrangement that is supplanted by the heterodimerization of YgjD with YeaZ in bacteria and heterodimerization of Kae1 with Pcc1 in KEOPS. The partial complementation of telomere maintenance by Qri7 hints that KEOPS has evolved novel functions in higher organisms.


Assuntos
Adenosina/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Adenosina/biossíntese , Adenosina/metabolismo , Dimerização , Metaloendopeptidases/fisiologia , Proteínas Mitocondriais/fisiologia , Modelos Moleculares , Subunidades Proteicas/fisiologia , RNA de Transferência/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/fisiologia , Homeostase do Telômero
9.
Mol Cell ; 45(3): 384-97, 2012 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-22325355

RESUMO

Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.


Assuntos
Cisteína Endopeptidases/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Proteínas Ubiquitinadas/metabolismo , Substituição de Aminoácidos , Linhagem Celular , Cristalografia por Raios X , Cisteína Endopeptidases/química , Cisteína Endopeptidases/genética , Enzimas Desubiquitinantes , Humanos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Organismos Geneticamente Modificados , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Quaternária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/genética , Ubiquitinação , Leveduras/genética , Leveduras/crescimento & desenvolvimento
10.
Mol Cell ; 40(4): 619-31, 2010 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-21055983

RESUMO

Genome integrity is jeopardized each time DNA replication forks stall or collapse. Here we report the identification of a complex composed of MMS22L (C6ORF167) and TONSL (NFKBIL2) that participates in the recovery from replication stress. MMS22L and TONSL are homologous to yeast Mms22 and plant Tonsoku/Brushy1, respectively. MMS22L-TONSL accumulates at regions of ssDNA associated with distressed replication forks or at processed DNA breaks, and its depletion results in high levels of endogenous DNA double-strand breaks caused by an inability to complete DNA synthesis after replication fork collapse. Moreover, cells depleted of MMS22L are highly sensitive to camptothecin, a topoisomerase I poison that impairs DNA replication progression. Finally, MMS22L and TONSL are necessary for the efficient formation of RAD51 foci after DNA damage, and their depletion impairs homologous recombination. These results indicate that MMS22L and TONSL are genome caretakers that stimulate the recombination-dependent repair of stalled or collapsed replication forks.


Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , NF-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Recombinação Genética , Estresse Fisiológico , Sobrevivência Celular , Quebras de DNA de Cadeia Dupla , Células HeLa , Humanos , NF-kappa B/química , Ligação Proteica , Fase S , Moldes Genéticos
11.
DNA Repair (Amst) ; 9(12): 1229-40, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-21056014

RESUMO

Protein ubiquitylation has emerged as an important regulatory mechanism that impacts almost every aspect of the DNA damage response. In this review, we discuss how DNA repair and checkpoint pathways utilize the diversity offered by the ubiquitin conjugation system to modulate the response to genotoxic lesions in space and time. In particular, we will highlight recent work done on the regulation of DNA double-strand breaks signalling and repair by the RNF8/RNF168 E3 ubiquitin ligases, the Fanconi anemia pathway and the role of protein degradation in the enforcement and termination of checkpoint signalling. We also discuss the various functions of deubiquitylating enzymes in these processes along with potential avenues for exploiting the ubiquitin conjugation/deconjugation system for therapeutic purposes.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Genes cdc/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais/genética
12.
Nat Struct Mol Biol ; 17(3): 299-305, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20139982

RESUMO

Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication-fork stalling results in phosphorylation of histone H2A yielding gamma-H2A (yeast gamma-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)-dependent manner. Here, we describe the genome-wide location analysis of gamma-H2A as a strategy to identify loci prone to engaging the Mec1 and Tel1 pathways. Notably, gamma-H2A enrichment overlaps with loci prone to replication-fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for gamma-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of gamma-H2A at these loci. Finally, our work indicates that high-resolution mapping of gamma-H2AX is a fruitful route to map fragile sites in eukaryotic genomes.


Assuntos
Genoma Fúngico/genética , Histonas/genética , Saccharomyces cerevisiae/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Imunoprecipitação da Cromatina , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosforilação , Reação em Cadeia da Polimerase , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
PLoS Biol ; 7(2): e41, 2009 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-19243221

RESUMO

The N-methyl-D-aspartate receptor (NMDAR), a major excitatory ligand-gated ion channel in the central nervous system (CNS), is a principal mediator of synaptic plasticity. Here we report that neuropilin tolloid-like 1 (Neto1), a complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane protein, is a novel component of the NMDAR complex critical for maintaining the abundance of NR2A-containing NMDARs in the postsynaptic density. Neto1-null mice have depressed long-term potentiation (LTP) at Schaffer collateral-CA1 synapses, with the subunit dependency of LTP induction switching from the normal predominance of NR2A- to NR2B-NMDARs. NMDAR-dependent spatial learning and memory is depressed in Neto1-null mice, indicating that Neto1 regulates NMDA receptor-dependent synaptic plasticity and cognition. Remarkably, we also found that the deficits in LTP, learning, and memory in Neto1-null mice were rescued by the ampakine CX546 at doses without effect in wild-type. Together, our results establish the principle that auxiliary proteins are required for the normal abundance of NMDAR subunits at synapses, and demonstrate that an inherited learning defect can be rescued pharmacologically, a finding with therapeutic implications for humans.


Assuntos
Aprendizagem/fisiologia , Lipoproteínas LDL/metabolismo , Proteínas de Membrana/metabolismo , Plasticidade Neuronal/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/genética , Animais , Linhagem Celular , Dioxóis/farmacologia , Hipocampo/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL , Aprendizagem/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/genética , Masculino , Memória/efeitos dos fármacos , Memória/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasticidade Neuronal/efeitos dos fármacos , Piperidinas/farmacologia , Transmissão Sináptica/efeitos dos fármacos
14.
Mol Cell ; 32(2): 259-75, 2008 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-18951093

RESUMO

Kae1 is a universally conserved ATPase and part of the essential gene set in bacteria. In archaea and eukaryotes, Kae1 is embedded within the protein kinase-containing KEOPS complex. Mutation of KEOPS subunits in yeast leads to striking telomere and transcription defects, but the exact biochemical function of KEOPS is not known. As a first step to elucidating its function, we solved the atomic structure of archaea-derived KEOPS complexes involving Kae1, Bud32, Pcc1, and Cgi121 subunits. Our studies suggest that Kae1 is regulated at two levels by the primordial protein kinase Bud32, which is itself regulated by Cgi121. Moreover, Pcc1 appears to function as a dimerization module, perhaps suggesting that KEOPS may be a processive molecular machine. Lastly, as Bud32 lacks the conventional substrate-recognition infrastructure of eukaryotic protein kinases including an activation segment, Bud32 may provide a glimpse of the evolutionary history of the protein kinase family.


Assuntos
Proteínas Arqueais/química , Complexos Multiproteicos/química , Proteínas Quinases/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Transporte/química , Cristalografia por Raios X , Escherichia coli/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Mathanococcus/genética , Mathanococcus/metabolismo , Modelos Moleculares , Complexos Multiproteicos/fisiologia , Ressonância Magnética Nuclear Biomolecular , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Homologia de Sequência de Aminoácidos , Telômero/metabolismo , Thermoplasma/genética , Thermoplasma/metabolismo , Transcrição Gênica
15.
PLoS Genet ; 3(8): e134, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17696614

RESUMO

Genome instability is a hallmark of cancer cells. One class of genome aberrations prevalent in tumor cells is termed gross chromosomal rearrangements (GCRs). GCRs comprise chromosome translocations, amplifications, inversions, deletion of whole chromosome arms, and interstitial deletions. Here, we report the results of a genome-wide screen in Saccharomyces cerevisiae aimed at identifying novel suppressors of GCR formation. The most potent novel GCR suppressor identified is BUD16, the gene coding for yeast pyridoxal kinase (Pdxk), a key enzyme in the metabolism of pyridoxal 5' phosphate (PLP), the biologically active form of vitamin B6. We show that Pdxk potently suppresses GCR events by curtailing the appearance of DNA lesions during the cell cycle. We also show that pharmacological inhibition of Pdxk in human cells leads to the production of DSBs and activation of the DNA damage checkpoint. Finally, our evidence suggests that PLP deficiency threatens genome integrity, most likely via its role in dTMP biosynthesis, as Pdxk-deficient cells accumulate uracil in their nuclear DNA and are sensitive to inhibition of ribonucleotide reductase. Since Pdxk links diet to genome stability, our work supports the hypothesis that dietary micronutrients reduce cancer risk by curtailing the accumulation of DNA damage and suggests that micronutrient depletion could be part of a defense mechanism against hyperproliferation.


Assuntos
Aberrações Cromossômicas , Cromossomos Fúngicos , Dano ao DNA , Genes Supressores , Fosfato de Piridoxal/fisiologia , Saccharomyces cerevisiae/genética , Quebras de DNA de Cadeia Dupla , Genes Supressores/fisiologia , Genes cdc , Técnicas Genéticas , Genoma Fúngico , Instabilidade Genômica , Células HeLa , Humanos , Modelos Biológicos , Piridoxal Quinase/genética , Piridoxal Quinase/fisiologia , Fosfato de Piridoxal/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Supressão Genética
16.
Curr Biol ; 16(14): R544-6, 2006 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-16860732

RESUMO

Telomeres are nucleoprotein structures that shelter the ends of linear chromosomes from being inappropriately recognized as DNA double-strand breaks. New work has revealed that Apollo, a nuclease previously implicated in DNA repair, also has a role in safeguarding telomeres during S phase.


Assuntos
Exodesoxirribonucleases/fisiologia , Proteínas Nucleares/fisiologia , Telômero/metabolismo , Reparo do DNA , Enzimas Reparadoras do DNA , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Subunidades Proteicas/metabolismo , Fase S , Complexo Shelterina , Proteínas Semelhantes à Proteína de Ligação a TATA-Box/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Proteína 2 de Ligação a Repetições Teloméricas
17.
Proc Natl Acad Sci U S A ; 101(6): 1754-9, 2004 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-14745032

RESUMO

Retinal bipolar cells are interneurons that transmit visual signals from photoreceptors to ganglion cells. Although the visual pathways mediated by bipolar cells have been well characterized, the genes that regulate their development and function are largely unknown. To determine the role in bipolar cell development of the homeobox gene Vsx1, whose retinal expression is restricted to a major subset of differentiating and mature cone bipolar (CB) cells, we targeted the gene in mice. Bipolar cell fate was not altered in the absence of Vsx1 function, because the pan-bipolar markers Chx10 and Ret-B1 continued to be expressed in inner nuclear layer neurons labeled by the Vsx1-targeting reporter gene, tauLacZ. The specification, number, and gross morphology of the subset of on-center and off-center (OFF)-CB cells defined by tauLacZ expression from the Vsx1 locus were also normal in Vsx1(tauLacZ)/Vsx1(tauLacZ) mice. However, the terminal differentiation of OFF-CB cells in the retina of Vsx1(tauLacZ)/Vsx1(tauLacZ) mice was incomplete, as demonstrated by a substantial reduction in the expression of at least four markers (recoverin, NK3R, Neto1, and CaB5) for these interneurons. These molecular abnormalities were associated with defects in retinal function and documented by electroretinography and in vitro ganglion cell recordings specific to cone visual signaling. In particular, there was a general reduction in the light-mediated activity of OFF, but not on-center, ganglion cells. Thus, Vsx1 is required for the late differentiation and function of OFF-CB cells and is associated with a heritable OFF visual pathway-specific retinal defect.


Assuntos
Diferenciação Celular , Proteínas do Olho/genética , Proteínas de Homeodomínio/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Transdução de Sinais , Animais , Camundongos , Células Fotorreceptoras Retinianas Cones/citologia
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