RESUMEN
We characterize the Polycomb system that assembles repressive subtelomeric domains of H3K27 methylation (H3K27me) in the yeast Cryptococcus neoformans. Purification of this PRC2-like protein complex reveals orthologs of animal PRC2 components as well as a chromodomain-containing subunit, Ccc1, which recognizes H3K27me. Whereas removal of either the EZH or EED ortholog eliminates H3K27me, disruption of mark recognition by Ccc1 causes H3K27me to redistribute. Strikingly, the resulting pattern of H3K27me coincides with domains of heterochromatin marked by H3K9me. Indeed, additional removal of the C. neoformans H3K9 methyltransferase Clr4 results in loss of both H3K9me and the redistributed H3K27me marks. These findings indicate that the anchoring of a chromatin-modifying complex to its product suppresses its attraction to a different chromatin type, explaining how enzymes that act on histones, which often harbor product recognition modules, may deposit distinct chromatin domains despite sharing a highly abundant and largely identical substrate-the nucleosome.
Asunto(s)
Cryptococcus neoformans/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas del Grupo Polycomb/metabolismo , Secuencia de Aminoácidos , Centrómero/metabolismo , Cryptococcus neoformans/genética , Heterocromatina/metabolismo , Código de Histonas , N-Metiltransferasa de Histona-Lisina/metabolismo , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
The entry of mammalian cells into the DNA synthesis phase (S phase) represents a key event in cell division1. According to current models of the cell cycle, the kinase CDC7 constitutes an essential and rate-limiting trigger of DNA replication, acting together with the cyclin-dependent kinase CDK2. Here we show that CDC7 is dispensable for cell division of many different cell types, as determined using chemical genetic systems that enable acute shutdown of CDC7 in cultured cells and in live mice. We demonstrate that another cell cycle kinase, CDK1, is also active during G1/S transition both in cycling cells and in cells exiting quiescence. We show that CDC7 and CDK1 perform functionally redundant roles during G1/S transition, and at least one of these kinases must be present to allow S-phase entry. These observations revise our understanding of cell cycle progression by demonstrating that CDK1 physiologically regulates two distinct transitions during cell division cycle, whereas CDC7 has a redundant function in DNA replication.
Asunto(s)
Proteínas de Ciclo Celular , Fase G1 , Proteínas Serina-Treonina Quinasas , Proteolisis , Fase S , Animales , Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Ratones , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Mammalian cells integrate mitogen and stress signalling before the end of G1 phase to determine whether or not they enter the cell cycle1-4. Before cells can replicate their DNA in S phase, they have to activate cyclin-dependent kinases (CDKs), induce an E2F transcription program and inactivate the anaphase-promoting complex (APC/CCDH1, also known as the cyclosome), which is an E3 ubiquitin ligase that contains the co-activator CDH1 (also known as FZR, encoded by FZR1). It was recently shown that stress can return cells to quiescence after CDK2 activation and E2F induction but not after inactivation of APC/CCDH1, which suggests that APC/CCDH1 inactivation is the point of no return for cell-cycle entry 3 . Rapid inactivation of APC/CCDH1 requires early mitotic inhibitor 1 (EMI1)3,5, but the molecular mechanism that controls this cell-cycle commitment step is unknown. Here we show using human cell models that cell-cycle commitment is mediated by an EMI1-APC/CCDH1 dual-negative feedback switch, in which EMI1 is both a substrate and an inhibitor of APC/CCDH1. The inactivation switch triggers a transition between a state with low EMI1 levels and high APC/CCDH1 activity during G1 and a state with high EMI1 levels and low APC/CCDH1 activity during S and G2. Cell-based analysis, in vitro reconstitution and modelling data show that the underlying dual-negative feedback is bistable and represents a robust irreversible switch. Our study suggests that mammalian cells commit to the cell cycle by increasing CDK2 activity and EMI1 mRNA expression to trigger a one-way APC/CCDH1 inactivation switch that is mediated by EMI1 transitioning from acting as a substrate of APC/CCDH1 to being an inhibitor of APC/CCDH1.
Asunto(s)
Proteínas Cdh1/antagonistas & inhibidores , Proteínas Cdh1/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Proteínas F-Box/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina E/metabolismo , Quinasa 2 Dependiente de la Ciclina/metabolismo , Proteínas F-Box/genética , Retroalimentación Fisiológica , Fase G1 , Células HeLa , Humanos , Fase SRESUMEN
Histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 9 trimethylation (H3K9me3) are epigenetic marks with opposing roles in transcription regulation. Whereas colocalization of these modifications is generally excluded in the genome, how this preclusion is established remains poorly understood. Lysine demethylase 4C (KDM4C), an H3K9me3 demethylase, localizes predominantly to H3K4me3-containing promoters through its hybrid tandem tudor domain (TTD) (1, 2), providing a model for how these modifications might be excluded. We quantitatively investigated the contribution of the TTD to the catalysis of H3K9me3 demethylation by KDM4C and demonstrated that TTD-mediated recognition of H3K4me3 stimulates demethylation of H3K9me3 in cis on peptide and mononucleosome substrates. Our findings support a multivalent interaction mechanism, by which an activating mark, H3K4me3, recruits and stimulates KDM4C to remove the repressive H3K9me3 mark, thus facilitating exclusion. In addition, our work suggests that differential TTD binding properties across the KDM4 demethylase family may differentiate their targets in the genome.
Asunto(s)
Cromatina/fisiología , Histonas/química , Histona Demetilasas con Dominio de Jumonji/química , Procesamiento Proteico-Postraduccional , Humanos , Cinética , Metilación , Nucleosomas/enzimología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Transducción de SeñalRESUMEN
Antiviral nucleoside analogues have been developed to inhibit the enzymatic activities of the hepatitis B virus (HBV) polymerase, thereby preventing the replication and production of HBV. However, the usage of these analogues can be limited by drug toxicity because the 5'-triphosphates of these nucleoside analogues (nucleotide analogues) are potential substrates for human DNA polymerases to incorporate into host DNA. Although they are poor substrates for human replicative DNA polymerases, it remains to be established whether these nucleotide analogues are substrates for the recently discovered human X- and Y-family DNA polymerases. Using presteady state kinetic techniques, we have measured the substrate specificity values for human DNA polymerases ß, λ, η, ι, κ, and Rev1 incorporating the active forms of the following anti-HBV nucleoside analogues approved for clinical use: adefovir, tenofovir, lamivudine, telbivudine, and entecavir. Compared to the incorporation of a natural nucleotide, most of the nucleotide analogues were incorporated less efficiently (2 to >122,000) by the six human DNA polymerases. In addition, the potential for entecavir and telbivudine, two drugs which possess a 3'-hydroxyl, to become embedded into human DNA was examined by primer extension and DNA ligation assays. These results suggested that telbivudine functions as a chain terminator, while entecavir was efficiently extended by the six enzymes and was a substrate for human DNA ligase I. Our findings suggested that incorporation of anti-HBV nucleotide analogues catalyzed by human X- and Y-family polymerases may contribute to clinical toxicity.
Asunto(s)
Antivirales/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/metabolismo , Nucleósidos/metabolismo , Purinas/metabolismo , Catálisis , Hepatitis B , Humanos , Cinética , Nucleótidos/metabolismoRESUMEN
Sulfolobus solfataricus DNA Polymerase IV (Dpo4), a prototype Y-family DNA polymerase, has been well characterized biochemically and biophysically at 37 °C or lower temperatures. However, the physiological temperature of the hyperthermophile S. solfataricus is approximately 80 °C. With such a large discrepancy in temperature, the in vivo relevance of these in vitro studies of Dpo4 has been questioned. Here, we employed circular dichroism spectroscopy and fluorescence-based thermal scanning to investigate the secondary structural changes of Dpo4 over a temperature range from 26 to 119 °C. Dpo4 was shown to display a high melting temperature characteristic of hyperthermophiles. Unexpectedly, the Little Finger domain of Dpo4, which is only found in the Y-family DNA polymerases, was shown to be more thermostable than the polymerase core. More interestingly, Dpo4 exhibited a three-state cooperative unfolding profile with an unfolding intermediate. The linker region between the Little Finger and Thumb domains of Dpo4 was found to be a source of structural instability. Through site-directed mutagenesis, the interactions between the residues in the linker region and the Palm domain were identified to play a critical role in the formation of the unfolding intermediate. Notably, the secondary structure of Dpo4 was not altered when the temperature was increased from 26 to 87.5 °C. Thus, in addition to providing structural insights into the thermal stability and an unfolding intermediate of Dpo4, our work also validated the relevance of the in vitro studies of Dpo4 performed at temperatures significantly lower than 80 °C.
Asunto(s)
ADN Polimerasa beta/metabolismo , Dicroismo Circular , ADN Polimerasa beta/química , ADN Polimerasa beta/genética , Colorantes Fluorescentes/química , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Desplegamiento Proteico , Sulfolobus solfataricus/enzimología , Temperatura de TransiciónRESUMEN
N-(Deoxyguanosin-8-yl)-1-aminopyrene (dG(AP)) is the predominant nitro polyaromatic hydrocarbon product generated from the air pollutant 1-nitropyrene reacting with DNA. Previous studies have shown that dG(AP) induces genetic mutations in bacterial and mammalian cells. One potential source of these mutations is the error-prone bypass of dG(AP) lesions catalyzed by the low-fidelity Y-family DNA polymerases. To provide a comparative analysis of the mutagenic potential of the translesion DNA synthesis (TLS) of dG(AP), we employed short oligonucleotide sequencing assays (SOSAs) with the model Y-family DNA polymerase from Sulfolobus solfataricus, DNA Polymerase IV (Dpo4), and the human Y-family DNA polymerases eta (hPolη), kappa (hPolκ), and iota (hPolι). Relative to undamaged DNA, all four enzymes generated far more mutations (base deletions, insertions, and substitutions) with a DNA template containing a site-specifically placed dG(AP). Opposite dG(AP) and at an immediate downstream template position, the most frequent mutations made by the three human enzymes were base deletions and the most frequent base substitutions were dAs for all enzymes. Based on the SOSA data, Dpo4 was the least error-prone Y-family DNA polymerase among the four enzymes during the TLS of dG(AP). Among the three human Y-family enzymes, hPolκ made the fewest mutations at all template positions except opposite the lesion site. hPolκ was significantly less error-prone than hPolι and hPolη during the extension of dG(AP) bypass products. Interestingly, the most frequent mutations created by hPolι at all template positions were base deletions. Although hRev1, the fourth human Y-family enzyme, could not extend dG(AP) bypass products in our standing start assays, it preferentially incorporated dCTP opposite the bulky lesion. Collectively, these mutagenic profiles suggest that hPolk and hRev1 are the most suitable human Y-family DNA polymerases to perform TLS of dG(AP) in humans.
Asunto(s)
Aductos de ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Mutágenos/toxicidad , Pirenos/toxicidad , Sulfolobus solfataricus/genéticaRESUMEN
Nucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antiviral drugs used to manage infections by human immunodeficiency virus, which causes AIDS. Unfortunately, these drugs cause unwanted side effects, and the molecular basis of NRTI toxicity is not fully understood. Putative routes of NRTI toxicity include the inhibition of human nuclear and mitochondrial DNA polymerases. A strong correlation between mitochondrial toxicity and NRTI incorporation catalyzed by human mitochondrial DNA polymerase has been established both in vitro and in vivo. However, it remains to be determined whether NRTIs are substrates for the recently discovered human X- and Y-family DNA polymerases, which participate in DNA repair and DNA lesion bypass in vivo. Using pre-steady-state kinetic techniques, we measured the substrate specificity constants for human DNA polymerases ß, λ, η, ι, κ, and Rev1 incorporating the active, 5'-phosphorylated forms of tenofovir, lamivudine, emtricitabine, and zidovudine. For the six enzymes, all of the drug analogs were incorporated less efficiently (40- to >110,000-fold) than the corresponding natural nucleotides, usually due to a weaker binding affinity and a slower rate of incorporation for the incoming nucleotide analog. In general, the 5'-triphosphate forms of lamivudine and zidovudine were better substrates than emtricitabine and tenofovir for the six human enzymes, although the substrate specificity profile depended on the DNA polymerase. Our kinetic results suggest NRTI insertion catalyzed by human X- and Y-family DNA polymerases is a potential mechanism of NRTI drug toxicity, and we have established a structure-function relationship for designing improved NRTIs.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Inhibidores de la Transcriptasa Inversa/química , Inhibidores de la Transcriptasa Inversa/metabolismo , Adenina/análogos & derivados , Adenina/química , Adenina/metabolismo , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Humanos , Lamivudine/química , Lamivudine/metabolismo , Estructura Molecular , Organofosfonatos/química , Organofosfonatos/metabolismo , Relación Estructura-Actividad , Tenofovir , Zidovudina/química , Zidovudina/metabolismoRESUMEN
Since their discovery as drivers of proliferation, cyclin-dependent kinases (CDKs) have been considered therapeutic targets. Small molecule inhibitors of CDK4/6 are used and tested in clinical trials to treat multiple cancer types. Despite their clinical importance, little is known about how CDK4/6 inhibitors affect the stability of CDK4/6 complexes, which bind cyclins and inhibitory proteins such as p21. We develop an assay to monitor CDK complex stability inside the nucleus. Unexpectedly, treatment with CDK4/6 inhibitors-palbociclib, ribociclib, or abemaciclib-immediately dissociates p21 selectively from CDK4 but not CDK6 complexes. This effect mediates indirect inhibition of CDK2 activity by p21 but not p27 redistribution. Our work shows that CDK4/6 inhibitors have two roles: non-catalytic inhibition of CDK2 via p21 displacement from CDK4 complexes, and catalytic inhibition of CDK4/6 independent of p21. By broadening the non-catalytic displacement to p27 and CDK6 containing complexes, next-generation CDK4/6 inhibitors may have improved efficacy and overcome resistance mechanisms.
Asunto(s)
Ciclina D/metabolismo , Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 6 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Animales , Ciclo Celular/efectos de los fármacos , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Humanos , Células MCF-7 , Ratones , Microscopía Fluorescente , Piperazinas/farmacología , Unión Proteica , Piridinas/farmacología , Epitelio Pigmentado de la Retina/citología , Epitelio Pigmentado de la Retina/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Mammalian cells typically start the cell-cycle entry program by activating cyclin-dependent protein kinase 4/6 (CDK4/6). CDK4/6 activity is clinically relevant as mutations, deletions, and amplifications that increase CDK4/6 activity contribute to the progression of many cancers. However, when CDK4/6 is activated relative to CDK2 remained incompletely understood. Here, we developed a reporter system to simultaneously monitor CDK4/6 and CDK2 activities in single cells and found that CDK4/6 activity increases rapidly before CDK2 activity gradually increases, and that CDK4/6 activity can be active after mitosis or inactive for variable time periods. Markedly, stress signals in G1 can rapidly inactivate CDK4/6 to return cells to quiescence but with reduced probability as cells approach S phase. Together, our study reveals a regulation of G1 length by temporary inactivation of CDK4/6 activity after mitosis, and a progressively increasing persistence in CDK4/6 activity that restricts cells from returning to quiescence as cells approach S phase.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/genética , Fase G1/genética , Estrés Fisiológico , Puntos de Control del Ciclo Celular , Línea Celular , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Genes Reporteros , Humanos , Mitosis , Fase S/genética , Análisis de la Célula Individual/métodosRESUMEN
Precise regulation of cellular proliferation is critical to tissue homeostasis and development, but misregulation leads to diseases of excess proliferation or cell loss. To achieve precise control, cells utilize distinct mechanisms of growth arrest such as quiescence and senescence. The decision to enter these growth-arrested states or proliferate is mediated by the core cell-cycle machinery that responds to diverse external and internal signals. Recent advances have revealed the molecular underpinnings of these cell-cycle decisions, highlighting the unique nature of cell-cycle entry from quiescence, identifying endogenous DNA damage as a quiescence-inducing signal, and establishing how persistent arrest is achieved in senescence.
Asunto(s)
Puntos de Control del Ciclo Celular , Ciclo Celular , Animales , Ciclo Celular/genética , Proliferación Celular , Senescencia Celular , Daño del ADN , Replicación del ADN , HumanosRESUMEN
The base excision repair (BER) pathway coordinates the replacement of 1-10 nucleotides at sites of single-base lesions. This process generates DNA substrates with various gap sizes which can alter the catalytic efficiency and fidelity of a DNA polymerase during gap-filling DNA synthesis. Here, we quantitatively determined the substrate specificity and base substitution fidelity of human DNA polymerase λ (Pol λ), an enzyme proposed to support the known BER DNA polymerase ß (Pol ß), as it filled 1-10-nucleotide gaps at 1-nucleotide intervals. Pol λ incorporated a correct nucleotide with relatively high efficiency until the gap size exceeded 9 nucleotides. Unlike Pol λ, Pol ß did not have an absolute threshold on gap size as the catalytic efficiency for a correct dNTP gradually decreased as the gap size increased from 2 to 10 nucleotides and then recovered for non-gapped DNA. Surprisingly, an increase in gap size resulted in lower polymerase fidelity for Pol λ, and this downregulation of fidelity was controlled by its non-enzymatic N-terminal domains. Overall, Pol λ was up to 160-fold more error-prone than Pol ß, thereby suggesting Pol λ would be more mutagenic during long gap-filling DNA synthesis. In addition, dCTP was the preferred misincorporation for Pol λ and its N-terminal domain truncation mutants. This nucleotide preference was shown to be dependent upon the identity of the adjacent 5'-template base. Our results suggested that both Pol λ and Pol ß would catalyze nucleotide incorporation with the highest combination of efficiency and accuracy when the DNA substrate contains a single-nucleotide gap. Thus, Pol λ, like Pol ß, is better suited to catalyze gap-filling DNA synthesis during short-patch BER in vivo, although, Pol λ may play a role in long-patch BER.
Asunto(s)
ADN Polimerasa beta/metabolismo , Reparación del ADN , ADN/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Bases , ADN/biosíntesis , ADN Polimerasa beta/química , ADN Polimerasa beta/genética , Desoxirribonucleótidos/metabolismo , Humanos , Cinética , Mutación , Especificidad por SustratoRESUMEN
DNA polymerase λ (Pol λ) is a novel X-family DNA polymerase that shares 34% sequence identity with DNA polymerase ß. Pre-steady-state kinetic studies have shown that the Pol λ-DNA complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the DNA polymerase ß-DNA complex, although the base substitution fidelity of both polymerases is 10(-)(4) to 10(-5). To better understand Pol λ's tight nucleotide binding affinity, we created single-substitution and double-substitution mutants of Pol λ to disrupt the interactions between active-site residues and an incoming nucleotide or a template base. Single-turnover kinetic assays showed that Pol λ binds to an incoming nucleotide via cooperative interactions with active-site residues (R386, R420, K422, Y505, F506, A510, and R514). Disrupting protein interactions with an incoming correct or incorrect nucleotide impacted binding to each of the common structural moieties in the following order: triphosphateâ«base>ribose. In addition, the loss of Watson-Crick hydrogen bonding between the nucleotide and the template base led to a moderate increase in K(d). The fidelity of Pol λ was maintained predominantly by a single residue, R517, which has minor groove interactions with the DNA template.
Asunto(s)
ADN Polimerasa beta/química , ADN Polimerasa beta/metabolismo , Regulación Alostérica , Sustitución de Aminoácidos , Emparejamiento Base , Secuencia de Bases , Dominio Catalítico/genética , ADN/química , ADN/genética , ADN/metabolismo , ADN Polimerasa beta/genética , Desoxirribonucleótidos/química , Desoxirribonucleótidos/metabolismo , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Cinética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
1-nitropyrene, the most abundant nitro polycyclic aromatic hydrocarbon in diesel emissions, has been found to react with DNA to form predominantly N-(deoxyguanosin-8-yl)-1-aminopyrene (dGAP). This bulky adduct has been shown to induce genetic mutations, which may implicate Y-family DNA polymerases in its bypass in vivo. To establish a kinetic mechanism for the bypass of such a prototype single-base lesion, we employed pre-steady-state kinetic methods to investigate individual nucleotide incorporations upstream, opposite, and downstream from a site-specifically placed dGAP lesion catalyzed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), a model Y-family DNA polymerase. Dpo4 was able to bypass dGAP but paused strongly at two sites: opposite the lesion and immediately downstream from the lesion. Both nucleotide incorporation efficiency and fidelity decreased significantly at the pause sites, especially during extension of the bypass product. Interestingly, a 4-fold tighter binding affinity of damaged DNA to Dpo4 promoted catalysis through putative interactions between the active site residues of Dpo4 and 1-aminopyrene moiety at the first pause site. In the presence of a DNA trap, the kinetics of nucleotide incorporation at these sites was biphasic in which a small, fast phase preceded a larger, slow phase. In contrast, only a large, fast phase was observed during nucleotide incorporation at non-pause sites. Our kinetic studies support a general kinetic mechanism for lesion bypass catalyzed by numerous DNA polymerases.