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1.
Mol Cell ; 79(5): 846-856.e8, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32755594

RESUMO

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.

2.
J Cell Sci ; 133(4)2020 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-32005696

RESUMO

USP16 (also known as UBP-M) has emerged as a histone H2AK119 deubiquitylase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also present in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has remained elusive. Here, we show that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for maintaining USP16 in the cytoplasm. We found that USP16 is only transiently retained in the nucleus following mitosis and then rapidly exported from this compartment. We also defined a non-canonical nuclear localization signal (NLS) sequence that plays a minimal role in directing USP16 into the nucleus. We further established that this DUB does not accumulate in the nucleus following DNA damage. Instead, only enforced nuclear localization of USP16 abolishes DNA double-strand break (DSB) repair, possibly due to unrestrained DUB activity. Thus, in contrast to the prevailing view, our data indicate that USP16 is actively excluded from the nucleus and that this DUB might indirectly regulate DSB repair.This article has an associated First Person interview with the first author of the paper.

3.
Elife ; 82019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30969169

RESUMO

The ability to isolate rare live cells within a heterogeneous population based solely on visual criteria remains technically challenging, due largely to limitations imposed by existing sorting technologies. Here, we present a new method that permits labeling cells of interest by attaching streptavidin-coated magnetic beads to their membranes using the lasers of a confocal microscope. A simple magnet allows highly specific isolation of the labeled cells, which then remain viable and proliferate normally. As proof of principle, we tagged, isolated, and expanded individual cells based on three biologically relevant visual characteristics: i) presence of multiple nuclei, ii) accumulation of lipid vesicles, and iii) ability to resolve ionizing radiation-induced DNA damage foci. Our method constitutes a rapid, efficient, and cost-effective approach for isolation and subsequent characterization of rare cells based on observable traits such as movement, shape, or location, which in turn can generate novel mechanistic insights into important biological processes.


Assuntos
Separação Celular/métodos , Campos Magnéticos , Coloração e Rotulagem/métodos , Estreptavidina/metabolismo , Animais , Linhagem Celular , Humanos
5.
DNA Repair (Amst) ; 74: 26-37, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30665830

RESUMO

DNA fiber fluorography is widely employed to study the kinetics of DNA replication, but the usefulness of this approach has been limited by the lack of freely-available automated analysis tools. Quantification of DNA fibers usually relies on manual examination of immunofluorescence microscopy images, which is laborious and prone to inter- and intra-operator variability. To address this, we developed an unbiased, fully automated algorithm that quantifies length and color of DNA fibers from fluorescence microscopy images. Our fiber quantification method, termed FiberQ, is an open-source image processing tool based on edge detection and a novel segment splicing approach. Here, we describe the algorithm in detail, validate our results experimentally, and benchmark the analysis against manual assessments. Our implementation is offered free of charge to the scientific community under the General Public License.


Assuntos
Algoritmos , DNA/química , Processamento de Imagem Assistida por Computador/métodos , Microscopia de Fluorescência , Fatores de Tempo
6.
Cancer Res ; 78(19): 5561-5573, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30072396

RESUMO

Intrinsic and acquired resistance to cisplatin remains a primary hurdle to treatment of high-grade serous ovarian cancer (HGSOC). Cisplatin selectively kills tumor cells by inducing DNA crosslinks that block replicative DNA polymerases. Single-stranded DNA (ssDNA) generated at resulting stalled replication forks (RF) is bound and protected by heterotrimeric replication protein A (RPA), which then serves as a platform for recruitment and activation of replication stress response factors. Cells deficient in this response are characterized by extensive ssDNA formation and excessive RPA recruitment that exhausts the available pool of RPA, which (i) inhibits RPA-dependent processes such as nucleotide excision repair (NER) and (ii) causes catastrophic failure of blocked RF. Here, we investigated the influence of RPA availability on chemosensitivity using a panel of human HGSOC cell lines. Our data revealed a striking correlation among these cell lines between cisplatin sensitivity and the inability to efficiently repair DNA via NER, specifically during S phase. Such defects in NER were attributable to RPA exhaustion arising from aberrant activation of DNA replication origins during replication stress. Reduced RPA availability promoted Mre11-dependent degradation of nascent DNA at stalled RF in cell lines exhibiting elevated sensitivity to cisplatin. Strikingly, defective S-phase NER, RF instability, and cisplatin sensitivity could all be rescued by ectopic overexpression of RPA. Taken together, our findings indicate that RPA exhaustion represents a major determinant of cisplatin sensitivity in HGSOC cell lines.Significance: The influence of replication protein A exhaustion on cisplatin sensitivity harbors important implications toward improving therapy of various cancers that initially respond to platinum-based agents but later relapse due to intrinsic or acquired drug resistance. Cancer Res; 78(19); 5561-73. ©2018 AACR.


Assuntos
Cisplatino/farmacologia , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Proteína de Replicação A/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , DNA de Cadeia Simples/genética , Feminino , Humanos , RNA Interferente Pequeno/metabolismo
7.
PLoS Genet ; 14(4): e1007356, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29659581

RESUMO

The evolutionarily-conserved sirtuin family of histone deacetylases regulates a multitude of DNA-associated processes. A recent genome-wide screen conducted in the yeast Saccharomyces cerevisiae identified Yku70/80, which regulate nonhomologous end-joining (NHEJ) and telomere structure, as being essential for cell proliferation in the presence of the pan-sirtuin inhibitor nicotinamide (NAM). Here, we show that sirtuin-dependent deacetylation of both histone H3 lysine 56 and H4 lysine 16 promotes growth of yku70Δ and yku80Δ cells, and that the NAM sensitivity of these mutants is not caused by defects in DNA double-strand break repair by NHEJ, but rather by their inability to maintain normal telomere length. Indeed, our results indicate that in the absence of sirtuin activity, cells with abnormally short telomeres, e.g., yku70/80Δ or est1/2Δ mutants, present striking defects in S phase progression. Our data further suggest that early firing of replication origins at short telomeres compromises the cellular response to NAM- and genotoxin-induced replicative stress. Finally, we show that reducing H4K16ac in yku70Δ cells limits activation of the DNA damage checkpoint kinase Rad53 in response to replicative stress, which promotes usage of translesion synthesis and S phase progression. Our results reveal a novel interplay between sirtuin-mediated regulation of chromatin structure and telomere-regulating factors in promoting timely completion of S phase upon replicative stress.


Assuntos
DNA Fúngico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sirtuínas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Reparo do DNA , Replicação do DNA , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genes Fúngicos , Histonas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Niacinamida/farmacologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Sirtuínas/antagonistas & inibidores , Sirtuínas/genética , Telomerase/genética , Telomerase/metabolismo , Telômero/genética , Telômero/metabolismo
8.
Front Microbiol ; 8: 1956, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29062309

RESUMO

Current antifungal drugs suffer from limitations including toxicity, the emergence of resistance and decreased efficacy at low pH that are typical of human vaginal surfaces. Here, we have shown that the antipsychotic drug valproic acid (VPA) exhibited a strong antifungal activity against both sensitive and resistant Candida albicans in pH condition similar to that encountered in vagina. VPA exerted a strong anti-biofilm activity and attenuated damage of vaginal epithelial cells caused by C. albicans. We also showed that VPA synergizes with the allylamine antifungal, Terbinafine. We undertook a chemogenetic screen to delineate biological processes that underlies VPA-sensitivity in C. albicans and found that vacuole-related genes were required to tolerate VPA. Confocal fluorescence live-cell imaging revealed that VPA alters vacuole integrity and support a model where alteration of vacuoles contributes to the antifungal activity. Taken together, this study suggests that VPA could be used as an effective antifungal against vulvovaginal candidiasis.

9.
J Cell Sci ; 130(15): 2579-2590, 2017 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-28600323

RESUMO

Retromer is a multimeric protein complex that mediates endosome-to-trans-Golgi network (TGN) and endosome-to-plasma membrane trafficking of integral membrane proteins. Dysfunction of this complex has been linked to Alzheimer's disease and Parkinson's disease. The recruitment of retromer to endosomes is regulated by Rab7 (also known as RAB7A) to coordinate endosome-to-TGN trafficking of cargo receptor complexes. Rab7 is also required for the degradation of internalized integral membrane proteins, such as the epidermal growth factor receptor (EGFR). We found that Rab7 is palmitoylated and that this modification is not required for membrane anchoring. Palmitoylated Rab7 colocalizes efficiently with and has a higher propensity to interact with retromer than nonpalmitoylatable Rab7. Rescue of Rab7 knockout cells by expressing wild-type Rab7 restores efficient endosome-to-TGN trafficking, while rescue with nonpalmitoylatable Rab7 does not. Interestingly, Rab7 palmitoylation does not appear to be required for the degradation of EGFR or for its interaction with its effector, Rab-interacting lysosomal protein (RILP). Overall, our results indicate that Rab7 palmitoylation is required for the spatiotemporal recruitment of retromer and efficient endosome-to-TGN trafficking of the lysosomal sorting receptors.


Assuntos
Endossomos/metabolismo , Lipoilação , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linhagem Celular , Endossomos/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , Rede trans-Golgi/genética
10.
Methods Mol Biol ; 1528: 149-164, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27854020

RESUMO

Antibodies that recognize specific histone modifications are invaluable tools to study chromatin structure and function. There are numerous commercially available antibodies that recognize a remarkable diversity of histone modifications. Unfortunately, many of them fail to work in certain applications or lack the high degree of specificity required of these reagents. The production of affinity-purified polyclonal antibodies against histone modifications demands a little effort but, in return, provides extremely valuable tools that overcome many of the concerns and limitations of commercial antibodies. We present a series of protocols and guidelines for the production and use of large amounts of polyclonal antibodies that recognize modifications of canonical histones. Our protocols can be applied to obtain antibodies that occur in histone variants and proteins other than histones. In addition, some of our protocols are compatible with the production of monoclonal or recombinant antibodies.


Assuntos
Histonas/metabolismo , Acetilação , Animais , Cromatina/metabolismo , Humanos , Metilação , Nucleossomos/metabolismo , Processamento de Proteína Pós-Traducional
11.
Sci Rep ; 6: 36013, 2016 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-27782169

RESUMO

The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes.


Assuntos
Ciclo Celular/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Saccharomyces cerevisiae/enzimologia , Ácido Valproico/farmacologia , Ciclo Celular/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Nucleic Acids Res ; 44(6): 2706-26, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26748095

RESUMO

The Saccharomyces cerevisiae genome encodes five sirtuins (Sir2 and Hst1-4), which constitute a conserved family of NAD-dependent histone deacetylases. Cells lacking any individual sirtuin display mild growth and gene silencing defects. However, hst3Δ hst4Δ double mutants are exquisitely sensitive to genotoxins, and hst3Δ hst4Δ sir2Δmutants are inviable. Our published data also indicate that pharmacological inhibition of sirtuins prevents growth of several fungal pathogens, although the biological basis is unclear. Here, we present genome-wide fitness assays conducted with nicotinamide (NAM), a pan-sirtuin inhibitor. Our data indicate that NAM treatment causes yeast to solicit specific DNA damage response pathways for survival, and that NAM-induced growth defects are mainly attributable to inhibition of Hst3 and Hst4 and consequent elevation of histone H3 lysine 56 acetylation (H3K56ac). Our results further reveal that in the presence of constitutive H3K56ac, the Slx4 scaffolding protein and PP4 phosphatase complex play essential roles in preventing hyperactivation of the DNA damage-response kinase Rad53 in response to spontaneous DNA damage caused by reactive oxygen species. Overall, our data support the concept that chromosome-wide histone deacetylation by sirtuins is critical to mitigate growth defects caused by endogenous genotoxins.


Assuntos
Cromatina/enzimologia , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Histonas/genética , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sirtuína 2/genética , Acetilação/efeitos dos fármacos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Cromatina/química , Cromatina/efeitos dos fármacos , Dano ao DNA , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Niacinamida/farmacologia , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Estresse Fisiológico
13.
J Biol Chem ; 291(2): 522-37, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26578521

RESUMO

Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase η (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1-3 overexpression rescues such deficiency in either ATR- or polymerase η-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response.


Assuntos
Reparo do DNA/genética , Mutação/genética , Fase S/genética , Estresse Fisiológico/genética , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Humanos , Mutagênicos/toxicidade , Fosforilação/efeitos dos fármacos , Dímeros de Pirimidina/metabolismo , Proteína de Replicação A/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/efeitos dos fármacos
14.
J Biol Chem ; 290(48): 28643-63, 2015 Nov 27.
Artigo em Inglês | MEDLINE | ID: mdl-26416890

RESUMO

The deubiquitinase (DUB) and tumor suppressor BAP1 catalyzes ubiquitin removal from histone H2A Lys-119 and coordinates cell proliferation, but how BAP1 partners modulate its function remains poorly understood. Here, we report that BAP1 forms two mutually exclusive complexes with the transcriptional regulators ASXL1 and ASXL2, which are necessary for maintaining proper protein levels of this DUB. Conversely, BAP1 is essential for maintaining ASXL2, but not ASXL1, protein stability. Notably, cancer-associated loss of BAP1 expression results in ASXL2 destabilization and hence loss of its function. ASXL1 and ASXL2 use their ASXM domains to interact with the C-terminal domain (CTD) of BAP1, and these interactions are required for ubiquitin binding and H2A deubiquitination. The deubiquitination-promoting effect of ASXM requires intramolecular interactions between catalytic and non-catalytic domains of BAP1, which generate a composite ubiquitin-binding interface (CUBI). Notably, the CUBI engages multiple interactions with ubiquitin involving (i) the ubiquitin carboxyl hydrolase catalytic domain of BAP1, which interacts with the hydrophobic patch of ubiquitin, and (ii) the CTD domain, which interacts with a charged patch of ubiquitin. Significantly, we identified cancer-associated mutations of BAP1 that disrupt the CUBI and notably an in-frame deletion in the CTD that inhibits its interaction with ASXL1/2 and DUB activity and deregulates cell proliferation. Moreover, we demonstrated that BAP1 interaction with ASXL2 regulates cell senescence and that ASXL2 cancer-associated mutations disrupt BAP1 DUB activity. Thus, inactivation of the BAP1/ASXL2 axis might contribute to cancer development.


Assuntos
Proliferação de Células , Neoplasias/metabolismo , Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Células HEK293 , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neoplasias/genética , Neoplasias/patologia , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Proteases Específicas de Ubiquitina/genética
15.
Epigenetics ; 10(8): 677-91, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26075789

RESUMO

O-GlcNAcylation is a posttranslational modification catalyzed by the O-Linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and reversed by O-GlcNAcase (OGA). Numerous transcriptional regulators, including chromatin modifying enzymes, transcription factors, and co-factors, are targeted by O-GlcNAcylation, indicating that this modification is central for chromatin-associated processes. Recently, OGT-mediated O-GlcNAcylation was reported to be a novel histone modification, suggesting a potential role in directly coordinating chromatin structure and function. In contrast, using multiple biochemical approaches, we report here that histone O-GlcNAcylation is undetectable in mammalian cells. Conversely, O-GlcNAcylation of the transcription regulators Host Cell Factor-1 (HCF-1) and Ten-Eleven Translocation protein 2 (TET2) could be readily observed. Our study raises questions on the occurrence and abundance of O-GlcNAcylation as a histone modification in mammalian cells and reveals technical complications regarding the detection of genuine protein O-GlcNAcylation. Therefore, the identification of the specific contexts in which histone O-GlcNAcylation might occur is still to be established.


Assuntos
Proteínas de Ligação a DNA/genética , Epigênese Genética , Histonas/genética , Fator C1 de Célula Hospedeira/genética , Proteínas Proto-Oncogênicas/genética , beta-N-Acetil-Hexosaminidases/genética , Acilação , Animais , Cromatina/genética , Glicosilação , Células HEK293 , Histonas/metabolismo , Humanos , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional/genética , beta-N-Acetil-Hexosaminidases/metabolismo
16.
Genetics ; 200(1): 185-205, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25786853

RESUMO

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56Ac) is present in newly synthesized histones deposited throughout the genome during DNA replication. The sirtuins Hst3 and Hst4 deacetylate H3K56 after S phase, and virtually all histone H3 molecules are K56 acetylated throughout the cell cycle in hst3∆ hst4∆ mutants. Failure to deacetylate H3K56 causes thermosensitivity, spontaneous DNA damage, and sensitivity to replicative stress via molecular mechanisms that remain unclear. Here we demonstrate that unlike wild-type cells, hst3∆ hst4∆ cells are unable to complete genome duplication and accumulate persistent foci containing the homologous recombination protein Rad52 after exposure to genotoxic drugs during S phase. In response to replicative stress, cells lacking Hst3 and Hst4 also displayed intense foci containing the Rfa1 subunit of the single-stranded DNA binding protein complex RPA, as well as persistent activation of DNA damage-induced kinases. To investigate the basis of these phenotypes, we identified histone point mutations that modulate the temperature and genotoxic drug sensitivity of hst3∆ hst4∆ cells. We found that reducing the levels of histone H4 lysine 16 acetylation or H3 lysine 79 methylation partially suppresses these sensitivities and reduces spontaneous and genotoxin-induced activation of the DNA damage-response kinase Rad53 in hst3∆ hst4∆ cells. Our data further suggest that elevated DNA damage-induced signaling significantly contributes to the phenotypes of hst3∆ hst4∆ cells. Overall, these results outline a novel interplay between H3K56Ac, H3K79 methylation, and H4K16 acetylation in the cellular response to DNA damage.


Assuntos
Dano ao DNA , Histona Desacetilases/metabolismo , Histonas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetilação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Cromatina/genética , Cromatina/metabolismo , Histona Desacetilases/genética , Metilação , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
17.
Mol Cell Biol ; 32(1): 154-72, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22025679

RESUMO

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes are not due to defects in base excision repair, defects in DNA damage tolerance, or a lack of Rad51 loading at sites of DNA damage. Our results argue that the acute sensitivity of H3K56ac-deficient cells to MMS and camptothecin stems from a failure to complete the repair of specific types of DNA lesions by recombination and/or from defects in the completion of DNA replication.


Assuntos
Dano ao DNA , Replicação do DNA , Histonas/metabolismo , Lisina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilação , Antineoplásicos/farmacologia , Camptotecina/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , DNA Fúngico/genética , DNA Fúngico/metabolismo , Histonas/genética , Hidroxiureia/farmacologia , Lisina/genética , Metanossulfonato de Metila/farmacologia , Mutagênicos/farmacologia , Mutação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
18.
Structure ; 19(2): 221-31, 2011 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-21256037

RESUMO

Yeast Rtt109 promotes nucleosome assembly and genome stability by acetylating K9, K27, and K56 of histone H3 through interaction with either of two distinct histone chaperones, Vps75 or Asf1. We report the crystal structure of an Rtt109-AcCoA/Vps75 complex revealing an elongated Vps75 homodimer bound to two globular Rtt109 molecules to form a symmetrical holoenzyme with a ∼12 Å diameter central hole. Vps75 and Rtt109 residues that mediate complex formation in the crystals are also important for Rtt109-Vps75 interaction and H3K9/K27 acetylation both in vitro and in yeast cells. The same Rtt109 residues do not participate in Asf1-mediated Rtt109 acetylation in vitro or H3K56 acetylation in yeast cells, demonstrating that Asf1 and Vps75 dictate Rtt109 substrate specificity through distinct mechanisms. These studies also suggest that Vps75 binding stimulates Rtt109 catalytic activity by appropriately presenting the H3-H4 substrate within the central cavity of the holoenzyme to promote H3K9/K27 acetylation of new histones before deposition.


Assuntos
Acetilcoenzima A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Acetilação , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Montagem e Desmontagem da Cromatina , Cristalografia por Raios X , Expressão Gênica , Instabilidade Genômica , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Histonas/genética , Humanos , Lisina/metabolismo , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutagênese Sítio-Dirigida , Ligação Proteica , Proteínas Recombinantes de Fusão , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato
19.
Nat Med ; 16(7): 774-80, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20601951

RESUMO

Candida albicans is a major fungal pathogen that causes serious systemic and mucosal infections in immunocompromised individuals. In yeast, histone H3 Lys56 acetylation (H3K56ac) is an abundant modification regulated by enzymes that have fungal-specific properties, making them appealing targets for antifungal therapy. Here we demonstrate that H3K56ac in C. albicans is regulated by the RTT109 and HST3 genes, which respectively encode the H3K56 acetyltransferase (Rtt109p) and deacetylase (Hst3p). We show that reduced levels of H3K56ac sensitize C. albicans to genotoxic and antifungal agents. Inhibition of Hst3p activity by conditional gene repression or nicotinamide treatment results in a loss of cell viability associated with abnormal filamentous growth, histone degradation and gross aberrations in DNA staining. We show that genetic or pharmacological alterations in H3K56ac levels reduce virulence in a mouse model of C. albicans infection. Our results demonstrate that modulation of H3K56ac is a unique strategy for treatment of C. albicans and, possibly, other fungal infections.


Assuntos
Antifúngicos/farmacologia , Candida albicans/enzimologia , Candida albicans/patogenicidade , Candidíase/enzimologia , Proteínas Fúngicas/metabolismo , Histona Acetiltransferases/metabolismo , Histona Desacetilases/metabolismo , Histonas/metabolismo , Acetilação , Animais , Candida albicans/efeitos dos fármacos , Candidíase/genética , Sobrevivência Celular , Sistemas de Liberação de Medicamentos , Proteínas Fúngicas/genética , Histona Acetiltransferases/genética , Histona Desacetilases/genética , Camundongos , Niacinamida/farmacologia , Virulência
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