RESUMO
Several replication-initiation proteins are assembled stepwise onto replicators to form pre-replicative complexes (pre-RCs) to license eukaryotic DNA replication. We performed a yeast functional proteomic screen and identified the Rix1 complex members (Ipi1p-Ipi2p/Rix1-Ipi3p) as pre-RC components and critical determinants of replication licensing and replication-initiation frequency. Ipi3p interacts with pre-RC proteins, binds chromatin predominantly at ARS sequences in a cell cycle-regulated and ORC- and Noc3p-dependent manner and is required for loading Cdc6p, Cdt1p and MCM onto chromatin to form pre-RC during the M-to-G1 transition and for pre-RC maintenance in G1 phase-independent of its role in ribosome biogenesis. Moreover, Ipi1p and Ipi2p, but not other ribosome biogenesis proteins Rea1p and Utp1p, are also required for pre-RC formation and maintenance, and Ipi1p, -2p and -3p are interdependent for their chromatin association and function in pre-RC formation. These results establish a new framework for the hierarchy of pre-RC proteins, where the Ipi1p-2p-3p complex provides a critical link between ORC-Noc3p and Cdc6p-Cdt1p-MCM in replication licensing.
Assuntos
Replicação do DNA , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fase G1/fisiologia , Proteína 1 de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Complexo de Reconhecimento de Origem/genética , Complexo de Reconhecimento de Origem/metabolismo , Proteoma , Origem de Replicação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
Gastric cancer is a heterogeneous disease with multiple environmental etiologies and alternative pathways of carcinogenesis. Beyond mutations in TP53, alterations in other genes or pathways account for only small subsets of the disease. We performed exome sequencing of 22 gastric cancer samples and identified previously unreported mutated genes and pathway alterations; in particular, we found genes involved in chromatin modification to be commonly mutated. A downstream validation study confirmed frequent inactivating mutations or protein deficiency of ARID1A, which encodes a member of the SWI-SNF chromatin remodeling family, in 83% of gastric cancers with microsatellite instability (MSI), 73% of those with Epstein-Barr virus (EBV) infection and 11% of those that were not infected with EBV and microsatellite stable (MSS). The mutation spectrum for ARID1A differs between molecular subtypes of gastric cancer, and mutation prevalence is negatively associated with mutations in TP53. Clinically, ARID1A alterations were associated with better prognosis in a stage-independent manner. These results reveal the genomic landscape, and highlight the importance of chromatin remodeling, in the molecular taxonomy of gastric cancer.
Assuntos
Exoma , Mutação , Proteínas Nucleares/genética , Neoplasias Gástricas/genética , Fatores de Transcrição/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ciclo Celular/genética , Montagem e Desmontagem da Cromatina , Proteínas de Ligação a DNA , Feminino , Genes Neoplásicos , Estudos de Associação Genética , Humanos , Junções Intercelulares , Masculino , Instabilidade de Microssatélites , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , Análise de Sequência de DNA , Transdução de Sinais , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/mortalidade , Adulto JovemRESUMO
The heterohexameric origin recognition complex (ORC) has been implicated in many cellular activities, including DNA replication, transcriptional control, heterochromatin assembly, centromere and telomere function, and so on. Here, we report a new function for ORC in mediating histone methylation. Using the yeast two-hybrid system, we identify a physical interaction between Orc2p and Spp1p, a member of the Set1 complex, and we demonstrate the interaction between the endogenous ORC and Spp1p by co-immunoprecipitation from yeast extracts. Furthermore, we find that Orc2p physically interacts with trimethylated histone 3 lysine 4 (H3K4) on chromatin by co-immunoprecipitation. Finally, we show that the trimethylation of H3K4 is decreased in orc2-1 cells and abolished in orc2-1, spp1Delta double mutants. Our data reveal a novel facet of ORC in mediating histone methylation in collaboration with Spp1p and demonstrate a connection between ORC and chromatin structure via the Set1 complex.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação Fúngica da Expressão Gênica , Histonas/química , Lisina/química , Complexo de Reconhecimento de Origem , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatina/química , Proteínas Fúngicas/metabolismo , Histona-Lisina N-Metiltransferase , Imunoprecipitação , Metilação , Modelos Biológicos , Plasmídeos/metabolismo , Saccharomyces cerevisiae/genética , Temperatura , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
It has been shown that the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) 38K (ac98) is required for nucleocapsid assembly. However, the exact role of 38K in nucleocapsid assembly remains unknown. In the present study, we investigated the relationship between 38K and the nucleocapsid. Western blotting using polyclonal antibodies raised against 38K revealed that 38K was expressed in the late phase of infection in AcMNPV-infected Spodoptera frugiperda cells and copurified with budded virus (BV) and occlusion-derived virus (ODV). Biochemical fractionation of BV and ODV into the nucleocapsid and envelope components followed by Western blotting showed that 38K was associated with the nucleocapsids. Immunoelectron microscopic analysis revealed that 38K was specifically localized to the nucleocapsids in infected cells and appeared to be distributed over the cylindrical capsid sheath of nucleocapsid. Yeast two-hybrid assays were performed to examine potential interactions between 38K and nine known nucleocapsid shell-associated proteins (PP78/83, PCNA, VP1054, FP25, VLF-1, VP39, BV/ODV-C42, VP80, and P24), three non-nucleocapsid shell-associated proteins (P6.9, PP31, and BV/ODV-E26), and itself. The results revealed that 38K interacted with the nucleocapsid proteins VP1054, VP39, VP80, and 38K itself. These interactions were confirmed by coimmunoprecipitation assays in vivo. These data demonstrate that 38K is a novel nucleocapsid protein and provide a rationale for why 38K is essential for nucleocapsid assembly.