RESUMO
Nucleotide metabolism fuels normal DNA replication and is also primarily targeted by the DNA replication checkpoint when replication stalls. To reveal a comprehensive interconnection between genome maintenance and metabolism, we analyzed the metabolomic changes upon replication stress in the budding yeast S. cerevisiae. We found that upon treatment of cells with hydroxyurea, glucose is rapidly diverted to the oxidative pentose phosphate pathway (PPP). This effect is mediated by the AMP-dependent kinase, SNF1, which phosphorylates the transcription factor Mig1, thereby relieving repression of the gene encoding the rate-limiting enzyme of the PPP. Surprisingly, NADPH produced by the PPP is required for efficient recruitment of replication protein A (RPA) to single-stranded DNA, providing the signal for the activation of the Mec1/ATR-Rad53/CHK1 checkpoint signaling kinase cascade. Thus, SNF1, best known as a central energy controller, determines a fast mode of replication checkpoint activation through a redox mechanism. These findings establish that SNF1 provides a hub with direct links to cellular metabolism, redox, and surveillance of DNA replication in eukaryotes.
Assuntos
Replicação do DNA , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/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 , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Glucose/genética , Glucose/metabolismo , Glicólise/fisiologia , Hidroxiureia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , NADP/metabolismo , Via de Pentose Fosfato , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Besides entrapping sister chromatids, cohesin drives other high-order chromosomal structural dynamics like looping, compartmentalization and condensation. ESCO2 acetylates a subset of cohesin so that cohesion must be established and only be established between nascent sister chromatids. How this process is precisely achieved remains unknown. Here, we report that GSK3 family kinases provide higher hierarchical control through an ESCO2 regulator, CRL4MMS22L. GSK3s phosphorylate Thr105 in MMS22L, resulting in homo-dimerization of CRL4MMS22L and ESCO2 during S phase as evidenced by single-molecule spectroscopy and several biochemical approaches. A single phospho-mimicking mutation on MMS22L (T105D) is sufficient to mediate their dimerization and rescue the cohesion defects caused by GSK3 or MMS22L depletion, whereas non-phosphorylable T105A exerts dominant-negative effects even in wildtype cells. Through cell fractionation and time-course measurements, we show that GSK3s facilitate the timely chromatin association of MMS22L and ESCO2 and subsequently SMC3 acetylation. The necessity of ESCO2 dimerization implicates symmetric control of cohesion establishment in eukaryotes.
Assuntos
Acetiltransferases , Cromátides , Proteínas Cromossômicas não Histona , Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Segregação de Cromossomos , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Nucleares/metabolismo , Fase S , Humanos , Linhagem Celular , Leveduras , Proteínas Cromossômicas não Histona/metabolismo , CoesinasRESUMO
Initiation of eukaryotic chromosome replication follows a spatiotemporal program. The current model suggests that replication origins compete for a limited pool of initiation factors. However, it remains to be answered how these limiting factors are preferentially recruited to early origins. Here, we report that Dbf4 is enriched at early origins through its interaction with forkhead transcription factors Fkh1 and Fkh2. This interaction is mediated by the Dbf4 C terminus and was successfully reconstituted in vitro. An interaction-defective mutant, dbf4ΔC, phenocopies fkh alleles in terms of origin firing. Remarkably, genome-wide replication profiles reveal that the direct fusion of the DNA-binding domain (DBD) of Fkh1 to Dbf4 restores the Fkh-dependent origin firing but interferes specifically with the pericentromeric origin activation. Furthermore, Dbf4 interacts directly with Sld3 and promotes the recruitment of downstream limiting factors. These data suggest that Fkh1 targets Dbf4 to a subset of noncentromeric origins to promote early replication in a manner that is reminiscent of the recruitment of Dbf4 to pericentromeric origins by Ctf19.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Origem de Replicação/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genoma Fúngico/genética , Mutação , Proteínas Nucleares/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Origem de Replicação/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Cohesin acetyltransferases ESCO1 and ESCO2 play a vital role in establishing sister chromatid cohesion. How ESCO1 and ESCO2 are controlled in a DNA replication-coupled manner remains unclear in higher eukaryotes. Here we show a critical role of CUL4-RING ligases (CRL4s) in cohesion establishment via regulating ESCO2 in human cells. Depletion of CUL4A, CUL4B or DDB1 subunits substantially reduces the normal cohesion efficiency. We also show that MMS22L, a vertebrate ortholog of yeast Mms22, is one of DDB1 and CUL4-associated factors (DCAFs) involved in cohesion. Several lines of evidence show selective interaction of CRL4s with ESCO2 through LxG motif, which is lost in ESCO1. Depletion of either CRL4s or ESCO2 causes a defect in SMC3 acetylation, which can be rescued by HDAC8 inhibition. More importantly, both CRL4s and PCNA act as mediators for efficiently stabilizing ESCO2 on chromatin and catalyzing SMC3 acetylation. Taken together, we propose an evolutionarily conserved mechanism in which CRL4s and PCNA promote ESCO2-dependent establishment of sister chromatid cohesion.
Assuntos
Acetiltransferases/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Culina/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Ubiquitina/genética , Acetilação , Proteínas de Ciclo Celular/genética , Linhagem Celular , Cromatina/genética , Células HEK293 , Humanos , Proteínas Nucleares/genética , CoesinasRESUMO
The S-phase checkpoint plays an essential role in regulation of the ribonucleotide reductase (RNR) activity to maintain the dNTP pools. How eukaryotic cells respond appropriately to different levels of replication threats remains elusive. Here, we have identified that a conserved GSK-3 kinase Mck1 cooperates with Dun1 in regulating this process. Deleting MCK1 sensitizes dun1Δ to hydroxyurea (HU) reminiscent of mec1Δ or rad53Δ. While Mck1 is downstream of Rad53, it does not participate in the post-translational regulation of RNR as Dun1 does. Mck1 phosphorylates and releases the Crt1 repressor from the promoters of DNA damage-inducible genes as RNR2-4 and HUG1. Hug1, an Rnr2 inhibitor normally silenced, is induced as a counterweight to excessive RNR. When cells suffer a more severe threat, Mck1 inhibits HUG1 transcription. Consistently, only a combined deletion of HUG1 and CRT1, confers a dramatic boost of dNTP levels and the survival of mck1Δdun1Δ or mec1Δ cells assaulted by a lethal dose of HU. These findings reveal the division-of-labor between Mck1 and Dun1 at the S-phase checkpoint pathway to fine-tune dNTP homeostasis.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica/fisiologia , Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Ciclo Celular/genética , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Quinase 3 da Glicogênio Sintase/genética , Hidroxiureia/toxicidade , Nucleotídeos/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Ribonucleotídeo Redutases/genética , Ribonucleotídeo Redutases/metabolismo , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Cohesin is a DNA-associated protein complex that forms a tripartite ring controlling sister chromatid cohesion, chromosome segregation and organization, DNA replication, and gene expression. Sister chromatid cohesion is established by the protein acetyltransferase Eco1, which acetylates two conserved lysine residues on the cohesin subunit Smc3 and thereby ensures correct chromatid separation in yeast (Saccharomyces cerevisiae) and other eukaryotes. However, the consequence of Eco1-catalyzed cohesin acetylation is unknown, and the exact nature of the cohesive state of chromatids remains controversial. Here, we show that self-interactions of the cohesin subunits Scc1/Rad21 and Scc3 occur in a DNA replication-coupled manner in both yeast and human cells. Using cross-linking MS-based and in vivo disulfide cross-linking analyses of purified cohesin, we show that a subpopulation of cohesin may exist as dimers. Importantly, upon temperature-sensitive and auxin-induced degron-mediated Eco1 depletion, the cohesin-cohesin interactions became significantly compromised, whereas deleting either the deacetylase Hos1 or the Eco1 antagonist Wpl1/Rad61 increased cohesin dimer levels by â¼20%. These results indicate that cohesin dimerizes in the S phase and monomerizes in mitosis, processes that are controlled by Eco1, Wpl1, and Hos1 in the sister chromatid cohesion-dissolution cycle. These findings suggest that cohesin dimerization is controlled by the cohesion cycle and support the notion that a double-ring cohesin model operates in sister chromatid cohesion.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas Nucleares/metabolismo , Multimerização Proteica/fisiologia , Fase S/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferases/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos Fúngicos/genética , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CoesinasRESUMO
The pre-initiation complex (pre-IC) has been proposed for two decades as an intermediate right before the maturation of the eukaryotic DNA replication fork. However, its existence and biochemical nature remain enigmatic. Here, through combining several enrichment strategies, we are able to isolate an endogenous dimeric CMG-containing complex (designated as d-CMG) distinct from traditional single CMG (s-CMG) and in vitro reconstituted dimeric CMG. D-CMG is assembled upon entry into the S phase and shortly matures into s-CMG/replisome, leading to the fact that only ~ 5% of the total CMG-containing complexes can be detected as d-CMG in vivo. Mass spectra reveal that RPA and DNA Pol α/primase co-purify with s-CMG, but not with d-CMG. Consistently, the former fraction is able to catalyze DNA unwinding and de novo synthesis, while the latter catalyzes neither. The two CMGs in d-CMG display flexibly orientated conformations under an electronic microscope. When DNA Pol α-primase is inactivated, d-CMG % rose up to 29%, indicating an incomplete pre-IC/fork transition. These findings reveal biochemical properties of the d-CMG/pre-IC and provide in vivo evidence to support the pre-IC/fork transition as a bona fide step in replication initiation.
Assuntos
Replicação do DNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , DNA Polimerase I/antagonistas & inibidores , DNA Polimerase I/metabolismo , DNA Primase/antagonistas & inibidores , DNA Primase/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dimerização , Microscopia Eletrônica , Proteínas Nucleares/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidoresRESUMO
Two sister chromatids must be held together by a cohesion process from their synthesis during S phase to segregation in anaphase. Despite its pivotal role in accurate chromosome segregation, how cohesion is established remains elusive. Here, we demonstrate that yeast Rtt101-Mms1, Cul4 family E3 ubiquitin ligases are stronger dosage suppressors of loss-of-function eco1 mutants than PCNA The essential cohesion reaction, Eco1-catalyzed Smc3 acetylation is reduced in the absence of Rtt101-Mms1. One of the adaptor subunits, Mms22, associates directly with Eco1. Point mutations (L61D/G63D) in Eco1 that abolish the interaction with Mms22 impair Smc3 acetylation. Importantly, an eco1LGpol30A251V double mutant displays additive Smc3ac reduction. Moreover, Smc3 acetylation and cohesion defects also occur in the mutants of other replication-coupled nucleosome assembly (RCNA) factors upstream or downstream of Rtt101-Mms1, indicating unanticipated cross talk between histone modifications and cohesin acetylation. These data suggest that fork-associated Cul4-Ddb1 E3s, together with PCNA, coordinate chromatin reassembly and cohesion establishment on the newly replicated sister chromatids, which are crucial for maintaining genome and chromosome stability.
Assuntos
Cromátides/fisiologia , Proteínas Culina/metabolismo , Nucleossomos/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromátides/genética , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas Culina/genética , Replicação do DNA , Período de Replicação do DNA , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Nucleossomos/genética , Mutação Puntual , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
BACKGROUND: Recent studies have implicated that members of the DICKKOPF (DKK) were causally involved in large number of human cancers. This study was designed to investigate the relationship between the genetic variations of DKK family genes and the risk of gastric cancer (GC). METHODS: Six SNPs (single nucleotide polymorphisms) of DKK family genes, including rs2241529 in DKK1, rs3733635, rs17037102 and rs419764 in DKK2, rs3206824 in DKK3 and rs2073664 in DKK4, were selected and genotyped by restriction fragment length polymorphism (RFLP) and TaqMan SNP genotyping methods in 409 GC cases and 554 cancer-free controls in the Han population in eastern China. RESULTS: None of the six SNPs achieved significant association with the overall GC risk and stratified analysis by age, gender, smoking status, drinking status, tumor location and pathological classification confirmed these non-significant associations. CONCLUSIONS: Our study indicated that the studied six SNPs of DKKs would not be the risk factors for GC in this Han Chinese population. Studies of larger population for different ethnicities will be needed to warrant our findings.
Assuntos
Variação Genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Gástricas/genética , Proteínas Adaptadoras de Transdução de Sinal , Povo Asiático/genética , Quimiocinas , Feminino , Predisposição Genética para Doença , Técnicas de Genotipagem , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Fatores de RiscoRESUMO
BACKGROUND: The origin of eukaryotic histone modification enzymes still remains obscure. RESULTS: Prototypic KMT4/Dot1 from Archaea targets chromatin proteins (Sul7d and Cren7) and shows increased activity on Sul7d, but not Cren7, in the presence of DNA. CONCLUSION: Promiscuous aKMT4 could be regulated by chromatin environment. SIGNIFICANCE: This study supports the prokaryotic origin model of eukaryotic histone methyltransferases and sheds light on chromatin dynamics in Archaea. Histone methylation is one of the major epigenetic modifications even in early diverging unicellular eukaryotes. We show that a widespread lysine methyltransferase from Archaea (aKMT4), bears striking structural and functional resemblance to the core of distantly related eukaryotic KMT4/Dot1. aKMT4 methylates a set of various proteins, including the chromatin proteins Sul7d and Cren7, and RNA exosome components. Csl4- and Rrp4-exosome complexes are methylated in different patterns. aKMT4 can self-methylate intramolecularly and compete with other proteins for the methyl group. Automethylation is inhibited by suitable substrates or DNA in a concentration-dependent manner. The automethylated enzyme shows relatively compromised activity. aKMT4-8A mutant with abrogated automethylation shows a more than 150% increase in methylation of substrates, suggesting a possible mechanism to regulate methyltransferase activity. More interestingly, methylation of Sul7d, but not Cren7, by aKMT4 is significantly enhanced by DNA. MS/MS and kinetic analysis further suggest that aKMT4 methylates Sul7d in the chromatin context. These data provide a clue to the possible regulation of aKMT4 activity by the local chromatin environment, albeit as a promiscuous enzyme required for extensive and variegated lysine methylation in Sulfolobus. This study supports the prokaryotic origin model of eukaryotic histone modification enzymes and sheds light on regulation of archaeal chromatin.
Assuntos
Proteínas Arqueais/química , Proteínas de Ligação a DNA/química , Proteínas Metiltransferases/química , Processamento de Proteína Pós-Traducional , Sulfolobus/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas Arqueais/genética , Cromatina/química , Sequência Conservada , DNA Arqueal/química , Metilação , Dados de Sequência Molecular , Proteínas Metiltransferases/genética , Especificidade por SubstratoRESUMO
BACKGROUND: The patients with hepatocellular carcinoma (HCC) have poor prognosis due to being diagnosed at late stage or recurrence following surgery. It's critical to identify effective biomarkers that can improve overall diagnosis and treatment of HCC. METHODS: We performed a meta-analysis of all relative studies reporting the clinicopathological significance of CDH1 hypermethylation in HCC by using Review Manager 5.2. A comprehensive literature search was performed in EMBASE, PubMed, Web of Science and Google Scholar databases. Kaplan Meier Plotter online database was used for the determination of correlation between CDH1 mRNA expression and overall survival in patients with HCC. Odds Ratios (OR) with 95% corresponding confidence intervals (CIs) were calculated. A total of 12 relevant studies were included in the meta-analysis with 981 patients. RESULTS: The positive rate of CDH1 hypermethylation was significantly higher in HCC than in normal liver tissue; and the pooled OR was 4.34 with 95% CI 2.50-7.56, P<0.00001. CDH1 promoter in HCC was more frequently hypermethylated compared to the group of chronic liver disease (CLD); OR was 4.83 with 95% CI 2.67-8.72, P<0.00001. However, the rate of CDH1 promoter hypermethylation was not correlated with different grades as well as stages. High CDH1 mRNA expression was significantly correlated to better overall survival in all 231 HCC patients compared to 133 HCC patients with low level CDH1 mRNA expression; HR was 0.6 with 95% CI 0.42-0.85, P=0.0034. CONCLUSION: In summary, CDH1 promoter hypermethylation is a risk factor and promising biomarker for HCC carcinogenesis and diagnosis, as well as a predictor of poor prognosis.
Assuntos
Adenocarcinoma , Neoplasias Esofágicas , Neoplasias Primárias Múltiplas , Neoplasias Gástricas , Humanos , Neoplasias Esofágicas/patologia , Neoplasias Esofágicas/terapia , Neoplasias Esofágicas/cirurgia , Estudos Retrospectivos , Neoplasias Gástricas/patologia , Neoplasias Gástricas/terapia , Neoplasias Gástricas/cirurgia , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Adenocarcinoma/cirurgia , Neoplasias Primárias Múltiplas/patologia , Neoplasias Primárias Múltiplas/terapia , Neoplasias Primárias Múltiplas/cirurgia , Masculino , Pessoa de Meia-Idade , Feminino , Idoso , Carcinoma de Células Escamosas/terapia , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/cirurgia , Carcinoma de Células Escamosas do Esôfago/terapia , Carcinoma de Células Escamosas do Esôfago/patologia , Carcinoma de Células Escamosas do Esôfago/cirurgia , Gastrectomia/métodos , Terapia CombinadaRESUMO
BACKGROUND: A satisfactory animal model of breast cancer metastasizing to bone is unavailable. In this study, we used human breast cancer stem-like cells and human bone to build a novel "human-source" model of human breast cancer skeletal metastasis. METHODS: Human breast cancer stem-like cells, the CD44+/CD24-/lower subpopulation, was separated and cultured. Before injection with the stem-like cells, mice were implanted with human bone in the right or left dorsal flanks. Animals in Groups A, B, and C were injected with 1 x 10(5), 1 x 10(6) human breast cancer stem-like cells, and 1 x 10(6) parental MDA-MB-231 cells, respectively. A positive control group (D) without implantation of human bone was also injected with 1 x 10(6) MDA-MB-231 cells. Immunohistochemistry was performed for determination of CD34, CD105, smooth muscle antibody, CD44, CD24, cytokine, CXC chemokine receptor-4 (CXCR4), and osteopontin (OPN). mRNA levels of CD44, CD24, CXCR4, and OPN in bone metastasis tissues were analyzed by real-time quantitative polymerase chain reaction (PCR). RESULTS: Our results demonstrated that cells in implanted human bones of group B, which received 1 x 10(6) cancer stem-like cells, stained strongly positive for CD44, CXCR4, and OPN, whereas those of other groups showed no or minimum staining. Moreover, group B had the highest incidence of human bone metastasis (77.8%, P = 0.0230) and no accompaniment of other tissue metastasis. The real-time PCR showed an increase of CD44, CXCR4, and OPN mRNA in metastatic bone tissues in group B compared with those of groups C and D, however the expression of CD24 mRNA in group B were the lowest. CONCLUSIONS: In the novel "human source" model of breast cancer, breast cancer stem-like cells demonstrated a higher human bone-seeking ability. Its mechanism might be related to the higher expressions of CD44, CXCR4, and OPN, and the lower expression of CD24 in breast cancer stem-like cells.
Assuntos
Neoplasias Ósseas/secundário , Neoplasias da Mama/patologia , Antígeno CD24/análise , Modelos Animais de Doenças , Receptores de Hialuronatos/análise , Células-Tronco Neoplásicas/patologia , Animais , Linhagem Celular Tumoral , Feminino , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Imuno-Histoquímica , Camundongos , Osteopontina/análise , Fenótipo , Receptores CXCR4/análiseRESUMO
DNA replication checkpoint (Mec1-Mrc1-Rad53 in budding yeast) is an evolutionarily conserved surveillance system to ensure proper DNA replication and genome stability in all eukaryotes. Compared to its well-known function as a mediator of replication checkpoint, the exact role of Mrc1 as a component of normal replication forks remains relatively unclear. In this study, we provide in vitro biochemical evidence to support that yeast Mrc1 is able to enhance the activity of DNA polymerase ϵ (Pol ϵ), the major leading strand replicase. Mrc1 can selectively bind avidly to primer/template DNA bearing a single-stranded region, but not to double-stranded DNA (dsDNA). Mutations of the lysine residues within basic patch 1 (BP1) compromise both DNA binding and polymerase stimulatory activities. Interestingly, Mrc1-3D, a mutant mimicking phosphorylation by the Hog1/MAPK kinase during the osmotic stress response, retains DNA binding but not polymerase stimulation. The stimulatory effect is also abrogated in Mrc1 purified from cells treated with hydroxyurea (HU), which elicits replication checkpoint activation. Taken together with previous findings, these results imply that under unperturbed condition, Mrc1 has a DNA synthesis stimulatory activity, which can be eliminated via Mrc1 phosphorylation in response to replication and/or osmotic stresses.
Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Fúngico/metabolismo , Fosforilação , Ligação ProteicaRESUMO
Background: Although oral hygiene and health have long been reported to be associated with increased risk of gastric cancer (GC), the direct relationship of oral microbes with the risk of GC have not been evaluated fully. We aimed to test whether tongue coating microbiome was associated with GC risk. Methods: Pyrosequencing of 16S rRNA gene of tongue coating microbiome was used in 57 newly diagnosed gastric adenocarcinomas and 80 healthy controls. Benjamini-Hochberg (BH) was applied for multiple comparison correction. Co-abundance group (CAGs) analysis was adopted. Results: We found that higher relative abundance of Firmicutes, and lower of Bacteroidetes were associated with increased risk of GC. In genus level, Streptococcus trended with a higher risk of GC, the four other genera (Neisseria, Prevotella, Prevotella7, and Porphyromonas) were found to have a decreased risk of GC. Different from overall GC and non-cardia cancer, Alloprevotella and Veillonella trended with the higher risk of cardia cancer. Finally, we analyzed the microbiota by determining CAGs and six clusters were identified. Except the Cluster 2 (mainly Streptococcus and Abiotrophia), the other clusters had an inverse association with GC. Of them, the Cluster 6 (mainly Prevotella and Prevotella7 etc) had a relatively good classification power with 0.76 of AUC. Conclusion: Microbiome in tongue coating may have potential guiding value for early detection and prevention of GC.
RESUMO
By hybridizing human adult testis cDNA microarrays with human adult and embryo testis cDNA probes, we identified a novel human testis gene, PINCH 2. PINCH 2 expression was 3.4-fold higher in adult than in fetal testis. The full length of its cDNA was 963 bp, with a 354-bp open reading frame (ORF), encoding a 117-amino acid protein. PINCH 2 was a splicing isoform of PINCH. It shared one exon, which encoded the LIM domain, with PINCH gene in human genome. Multitissue reverse transcriptase-polymerase chain reaction (RTPCR) analysis revealed that this gene was expressed variably in a wide range of tissues, with high expression levels in human adult testis. These results suggest that PINCH 2, a novel LIM domain-containing gene, may play an important role in testicular development/spermatogenesis.
Assuntos
Proteínas de Ligação a DNA/genética , Testículo/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Processamento Alternativo , Sequência de Aminoácidos , Sequência de Bases , Biotecnologia , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Variação Genética , Humanos , Proteínas com Domínio LIM , Masculino , Proteínas de Membrana , Dados de Sequência Molecular , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Gravidez , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Espermatogênese/genética , Testículo/embriologia , Testículo/crescimento & desenvolvimento , Distribuição TecidualRESUMO
BACKGROUND: Secreted frizzled-related protein 1 (sFRP1), a negative regulator of the Wnt signaling pathway, is frequently inactivated in human gastric cancer. Genetic variants in the 3' untranslated region (UTR) of the gene may influence the strength of miRNA binding and the regulation of mRNA transcription, affecting the individual's cancer risk. This study aims to investigate the impact of variants in the 3' UTR of sFRP1 on the gastric cancer susceptibility in a Chinese population. PATIENTS AND METHODS: The association between 2 sFRP1 gene variation loci (rs1127379 and rs10088390) with minor allele frequency more than 0.1 in the 3' UTR and gastric cancer risk was assessed in a case-control study including 419 gastric cancer cases and 571 healthy controls. PCR-restriction fragment length polymorphism analysis was used for genotyping; the odds ratio and 95% confidence interval were calculated to estimate the relative risk. RESULTS: Compared with the AA genotype, the GG genotype of rs1127379 was significantly associated with a reduced risk of gastric cancer overall. In the subgroup analysis, the protective effect of the GG genotype was also found for noncardia cancer and intestinal gastric cancer. Furthermore, haplotype analysis showed that the A rs1127379 G rs10088390 haplotype conferred a risk effect for gastric cancer. CONCLUSIONS: Genetic variants at the sFRP1 gene may be involved in gastric tumorigenesis, especially in noncardia and intestinal gastric cancer. Further prospective studies with different ethnicities and large sample sizes are needed to confirm our findings.
Assuntos
Regiões 3' não Traduzidas/genética , Biomarcadores Tumorais/genética , Predisposição Genética para Doença , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Membrana/genética , Polimorfismo de Nucleotídeo Único/genética , Neoplasias Gástricas/etiologia , Estudos de Casos e Controles , China/epidemiologia , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Prognóstico , Neoplasias Gástricas/epidemiologia , Taxa de SobrevidaRESUMO
Sld3/Treslin is an evolutionarily conserved protein essential for activation of DNA helicase Mcm2-7 and replication initiation in all eukaryotes. Nevertheless, it remains elusive how Sld3 is recruited to origins. Here, we have identified the direct physical association of Sld3 with Mcm2 and Mcm6 subunits in vitro, which is significantly enhanced by DDK in vivo. The Sld3-binding domain (SBD) is mapped to the N-termini of Mcm2 and Mcm6, both of them are essential for cell viability and enriched with the DDK phosphorylation sites. Glutamic acid substitution of four conserved positively charged residues of Sld3 (sld3-4E), near the Cdc45-binding region, interrupts its interaction with Mcm2/6 and causes cell death. By using a temperature-inducible degron (td), we show that deletion of Mcm6 SBD (mcm6ΔN122) abolishes not only Sld3 enrichment at early origins in G1 phase, but also subsequent recruitment of GINS and RPA during S phase. These findings elucidate the in vivo molecular details of the DDK-dependent Sld3-MCM association, which plays a crucial role in MCM helicase activation and origin unwinding.
RESUMO
Methanogenesis and its key small-molecule methyltransferase Mtr complex are poorly understood despite their pivotal role in Earth's global carbon cycle. Mtr complex is encoded by a conserved mtrEDCBAFGH operon in most methanogens. Here we report that two discrete lineages, Methanococcales and Methanomicrobiales, have a noncanonical mtr operon carrying two copies of mtrA resulting from an ancient duplication. Compared to mtrA-1, mtrA-2 acquires a distinct transmembrane domain through domain shuffling and gene fusion. However, the nontransmembrane domains (MtrA domain) of mtrA-1 and mtrA-2 are homogenized by gene conversion events lasting throughout the long history of these extant methanogens (over 2410 million years). Furthermore, we identified a possible recruitment of ancient nonmethanogenic methyltransferase genes to establish the methanogenesis pathway. These results not only provide novel evolutionary insight into the methanogenesis pathway and methyltransferase superfamily but also suggest an unanticipated long-lasting effect of gene conversion on gene evolution in a convergent pattern.
Assuntos
Proteínas Arqueais/genética , Evolução Molecular , Conversão Gênica , Methanococcales/genética , Methanomicrobiales/genética , Metiltransferases/metabolismo , Sequência de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , Genes Arqueais , Metiltransferases/química , Metiltransferases/genética , Dados de Sequência MolecularRESUMO
BACKGROUND: The protective role of IL-11, an IL-6 family cytokine, has been implicated in ischemia/reperfusion injury (IRI) in the heart and kidney, but its role has not been elucidated in liver IRI. This study was designed to evaluate the effects of IL-11 and its mechanism of action on liver IRI in a mouse model. METHODS: A partial (70%) warm liver IRI was induced by interrupting the artery/portal vein blood supply to the left/middle liver lobes. IL-11 mRNA expression of ischemic liver after reperfusion was analyzed. Signal transducer and activator of transcription 3 (STAT3) was analyzed following IL-11 treatment in vivo and in vitro. Next, IL-11 was injected intraperitoneally (ip) 1 hour before ischemia. Liver injury was assessed based on serum alanine aminotransferase levels and histopathology. Apoptosis and inflammation were also determined in the ischemic liver. To analyze the role of STAT3 in IL-11 treatment, STAT3 siRNA or non-specific (NS) siRNA was used in vitro and in vivo. RESULTS: IL-11 mRNA expression was significantly increased after reperfusion in the ischemic liver. STAT3, as a target of IL-11, was activated in hepatocytes after IL-11 treatment in vivo and in vitro. Next, effects of IL-11/STAT3 signaling pathway were assessed in liver IRI, which showed IL-11 treatment significantly attenuated liver IRI, as evidenced by reduced hepatocellular function and hepatocellular necrosis/apoptosis. In addition, IL-11 treatment significantly inhibited the gene expressions of pro-inflammatory cytokines (TNF-α and IL-10) and chemokines (IP-10 and MCP-1). To determine the role of STAT3 in the hepatoprotective effects of IL-11, STAT3 siRNA or NS siRNA was used prior to IL-11 treatment. The results showed STAT3 knockdown abrogated the protective effects of IL-11 in vitro and in vivo. CONCLUSIONS: This work provides first-time evidence for the protective effect of IL-11 treatment on hepatocyte in liver IRI, through the activation of the STAT3 pathway.