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1.
Anticancer Res ; 39(7): 3565-3570, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262880

RESUMO

BACKGROUND/AIM: Trifluridine (FTD) is a key component of the novel oral antitumor drug trifluridine/tipiracil that has been approved for the treatment of metastatic colorectal cancer. In this study, a comprehensive analysis of DNA replication profile in FTD-treated colon cancer cells was performed. MATERIALS AND METHODS: HCT-116 cells were exposed to BrdU or FTD and subjected to DNA immunoprecipitation. Immunoprecipitated DNA was sequenced; the density of aligned reads along the genome was calculated. Peak finding, gene ontology, and motif analysis were performed using MACS, GREAT, and MEME, respectively. RESULTS: We identified 6,043 and 5,080 high-confidence FTD and BrdU peaks in HCT-116 cells, respectively. Of 6,043 FTD peaks, 2,911 peaks were uncommon to BrdU. We observed that FTD was preferentially incorporated into genomic regions containing simple repeats, CpG islands, and gene bodies. Conserved motifs in FTD peaks contained dinucleotide repeats such as (GT)n. CONCLUSION: Global FTD incorporation patterns delineated FTD, preferentially incorporating loci in cancer cells.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Bromodesoxiuridina/farmacologia , Neoplasias Colorretais/genética , Replicação do DNA/efeitos dos fármacos , Trifluridina/farmacologia , Células HCT116 , Humanos , Análise de Sequência de DNA
2.
J Cell Biol ; 218(7): 2080-2081, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31266845

RESUMO

The full licensing of replication origins in late G1 is normally enforced by the licensing checkpoint. In this issue, Matson et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201902143) show that this checkpoint is inoperative in cells exiting from G0, resulting in incomplete origin licensing and consequent replicative stress.


Assuntos
Replicação do DNA , Origem de Replicação , Ciclo Celular , Proteínas de Ciclo Celular , Divisão Celular
4.
Nat Commun ; 10(1): 2529, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175278

RESUMO

Substitution of lysine 27 with methionine in histone H3.3 is a recently discovered driver mutation of pediatric high-grade gliomas. Mutant cells show decreased levels and altered distribution of H3K27 trimethylation (H3K27me3). How these chromatin changes are established genome-wide and lead to tumorigenesis remains unclear. Here we show that H3.3K27M-mediated alterations in H3K27me3 distribution result in ectopic DNA replication and cell cycle progression of germ cells in Caenorhabditis elegans. By genetically inducing changes in the H3.3 distribution, we demonstrate that both H3.3K27M and pre-existing H3K27me3 act locally and antagonistically on Polycomb Repressive Complex 2 (PRC2) in a concentration-dependent manner. The heterochromatin changes result in extensive gene misregulation, and genetic screening identified upregulation of JNK as an underlying cause of the germcell aberrations. Moreover, JNK inhibition suppresses the replicative fate in human tumor-derived H3.3K27M cells, thus establishing C. elegans as a powerful model for the identification of potential drug targets for treatment of H3.3K27M tumors.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Ciclo Celular , Replicação do DNA , Regulação da Expressão Gênica , Histonas/metabolismo , Sistema de Sinalização das MAP Quinases , Animais , Neoplasias Encefálicas , Caenorhabditis elegans , Carcinogênese , Cromatina , Regulação Neoplásica da Expressão Gênica , Células Germinativas/metabolismo , Glioma , Heterocromatina , Código das Histonas , Metilação , Complexo Repressor Polycomb 2/metabolismo
5.
Nat Commun ; 10(1): 2502, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31175280

RESUMO

Accumulation of nucleotide building blocks prior to and during S phase facilitates DNA duplication. Herein, we find that the anaphase-promoting complex/cyclosome (APC/C) synchronizes ribose-5-phosphate levels and DNA synthesis during the cell cycle. In late G1 and S phases, transketolase-like 1 (TKTL1) is overexpressed and forms stable TKTL1-transketolase heterodimers that accumulate ribose-5-phosphate. This accumulation occurs by asymmetric production of ribose-5-phosphate from the non-oxidative pentose phosphate pathway and prevention of ribose-5-phosphate removal by depleting transketolase homodimers. In the G2 and M phases after DNA synthesis, expression of the APC/C adaptor CDH1 allows APC/CCDH1 to degrade D-box-containing TKTL1, abrogating ribose-5-phosphate accumulation by TKTL1. TKTL1-overexpressing cancer cells exhibit elevated ribose-5-phosphate levels. The low CDH1 or high TKTL1-induced accumulation of ribose-5-phosphate facilitates nucleotide and DNA synthesis as well as cell cycle progression in a ribose-5-phosphate-saturable manner. Here we reveal that the cell cycle control machinery regulates DNA synthesis by mediating ribose-5-phosphate sufficiency.


Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Proteínas Cdh1/metabolismo , Ciclo Celular , Replicação do DNA , Ribosemonofosfatos/metabolismo , Transcetolase/metabolismo , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Fase G2 , Humanos , Via de Pentose Fosfato , Fase S
6.
Nat Commun ; 10(1): 2420, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160570

RESUMO

Replication-Factor-C (RFC) and RFC-like complexes (RLCs) mediate chromatin engagement of the proliferating cell nuclear antigen (PCNA). It remains controversial how RFC and RLCs cooperate to regulate PCNA loading and unloading. Here, we show the distinct PCNA loading or unloading activity of each clamp loader. ATAD5-RLC possesses the potent PCNA unloading activity. ATPase motif and collar domain of ATAD5 are crucial for the unloading activity. DNA structures did not affect PCNA unloading activity of ATAD5-RLC. ATAD5-RLC could unload ubiquitinated PCNA. Through single molecule measurements, we reveal that ATAD5-RLC unloaded PCNA through one intermediate state before ATP hydrolysis. RFC loaded PCNA through two intermediate states on DNA, separated by ATP hydrolysis. Replication proteins such as Fen1 could inhibit the PCNA unloading activity of Elg1-RLC, a yeast homolog of ATAD5-RLC in vitro. Our findings provide molecular insights into how PCNA is released from chromatin to finalize DNA replication/repair.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína de Replicação C/metabolismo , Adenosina Trifosfatases , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Cromatina/metabolismo , Endonucleases Flap/metabolismo , Humanos , Hidrólise , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Nat Cell Biol ; 21(6): 669-671, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160706
8.
Genome Biol ; 20(1): 111, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31146781

RESUMO

Recent studies have accomplished the extraordinary feat of measuring the exact status of DNA replication in individual cells. We outline how these studies have revealed surprising uniformity in how cells replicate their DNA, and consider the implications of this remarkable technological advance.


Assuntos
Replicação do DNA , Análise de Célula Única , Genômica
9.
Nat Commun ; 10(1): 2426, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31160578

RESUMO

DNA replication initiation is a two-step process. During the G1-phase of the cell cycle, the ORC complex, CDC6, CDT1, and MCM2-7 assemble at replication origins, forming pre-replicative complexes (pre-RCs). In S-phase, kinase activities allow fork establishment through (CDC45/MCM2-7/GINS) CMG-complex formation. However, only a subset of all potential origins becomes activated, through a poorly understood selection mechanism. Here we analyse the pre-RC proteomic interactome in human cells and find C13ORF7/RNF219 (hereafter called OBI1, for ORC-ubiquitin-ligase-1) associated with the ORC complex. OBI1 silencing result in defective origin firing, as shown by reduced CMG formation, without affecting pre-RC establishment. OBI1 catalyses the multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase. Importantly, expression of non-ubiquitylable ORC3/5 mutants impairs origin firing, demonstrating their relevance as OBI1 substrates for origin firing. Our results identify a ubiquitin signalling pathway involved in origin activation and provide a candidate protein for selecting the origins to be fired.


Assuntos
Replicação do DNA/fisiologia , Fase G1/fisiologia , Complexo de Reconhecimento de Origem/metabolismo , Origem de Replicação/fisiologia , Fase S/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Humanos , Complexo de Reconhecimento de Origem/genética , Proteômica , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
10.
Nat Cell Biol ; 21(6): 743-754, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31160708

RESUMO

Chromatin assembled with the histone H3 variant CENP-A is the heritable epigenetic determinant of human centromere identity. Using genome-wide mapping and reference models for 23 human centromeres, CENP-A binding sites are identified within the megabase-long, repetitive α-satellite DNAs at each centromere. CENP-A is shown in early G1 to be assembled into nucleosomes within each centromere and onto 11,390 transcriptionally active sites on the chromosome arms. DNA replication is demonstrated to remove ectopically loaded, non-centromeric CENP-A. In contrast, tethering of centromeric CENP-A to the sites of DNA replication through the constitutive centromere associated network (CCAN) is shown to enable precise reloading of centromere-bound CENP-A onto the same DNA sequences as in its initial prereplication loading. Thus, DNA replication acts as an error correction mechanism for maintaining centromere identity through its removal of non-centromeric CENP-A coupled with CCAN-mediated retention and precise reloading of centromeric CENP-A.


Assuntos
Proteína Centromérica A/genética , Centrômero/genética , Cromossomos Humanos/genética , Replicação do DNA/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Fase G1/genética , Células HeLa , Histonas/genética , Humanos , Nucleossomos/genética
11.
BMC Complement Altern Med ; 19(1): 112, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31151442

RESUMO

BACKGROUND: The plant Holarrhena floribunda (H. floribunda; G. Don) is indigenous to sub-Saharan Africa and is traditionally used to treat several ailments. The present study was carried out to isolate and characterize bioactive compounds with anti-proliferative activity present in H. floribunda extracts. METHODS: Compounds were isolated from H. floribunda using the bioassay-guided fractionation technique of repeated column chromatography and the step-wise application of the MTT reduction assay to assess antiproliferative bioactivity. The structures of the compounds were identified mainly using NMR. The effects of the isolated compounds on the viability, cell cycle and proliferation of human cancer cell lines (MCF-7, HeLa and HT-29) as well as the non-cancerous human fibroblast cell line (KMST-6) were investigated. RESULTS: Bioassay-guided fractionation yielded two steroidal alkaloids: holamine (1) and funtumine (2). The MTT reduction assay shows that both compounds exhibited selective dose-dependent cytotoxicity against the cancer cell lines studied. The isolated compounds induced cell cycle arrest at the G0/G1 and G2/M phases in the cancer cell lines with significant reduction in DNA synthesis. The results obtained show that the cancer cells (MCF-7, HeLa and HT-29) used in this study were more sensitive to the isolated compounds compared to the noncancerous fibroblast cells (KMST-6). CONCLUSION: The ability of the isolated compounds to cause cell cycle arrest and reduce DNA synthesis raises hopes for their possible development and use as potent anticancer drugs. However, more mechanistic studies need to be done for complete validation of the efficacy of the two compounds.


Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Ciclo Celular/efeitos dos fármacos , Holarrhena/química , Fitosteróis/isolamento & purificação , Linhagem Celular Tumoral , Replicação do DNA/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Fitosteróis/farmacologia
12.
Nat Commun ; 10(1): 2194, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097704

RESUMO

Although the physical properties of chromosomes, including their morphology, mechanics, and dynamics are crucial for their biological function, many basic questions remain unresolved. Here we directly image the circular chromosome in live E. coli with a broadened cell shape. We find that it exhibits a torus topology with, on average, a lower-density origin of replication and an ultrathin flexible string of DNA at the terminus of replication. At the single-cell level, the torus is strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. Our data show a domain organization underlying the chromosome structure of E. coli, where MatP proteins induce site-specific persistent domain boundaries at Ori/Ter, while transcription regulators HU and Fis induce weaker transient domain boundaries throughout the genome. These findings provide an architectural basis for the understanding of the dynamic spatial organization of bacterial genomes in live cells.


Assuntos
Cromossomos Bacterianos/química , DNA Bacteriano/química , DNA Circular/química , Escherichia coli/genética , Genoma Bacteriano , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos Bacterianos/metabolismo , Replicação do DNA , DNA Bacteriano/metabolismo , DNA Circular/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Microscopia Intravital/instrumentação , Microscopia Intravital/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Conformação de Ácido Nucleico , Análise de Célula Única/instrumentação , Análise de Célula Única/métodos
13.
Nat Commun ; 10(1): 2313, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127121

RESUMO

DNA double-strand breaks (DSBs) are among the most lethal types of DNA damage and frequently cause genome instability. Sequencing-based methods for mapping DSBs have been developed but they allow measurement only of relative frequencies of DSBs between loci, which limits our understanding of the physiological relevance of detected DSBs. Here we propose quantitative DSB sequencing (qDSB-Seq), a method providing both DSB frequencies per cell and their precise genomic coordinates. We induce spike-in DSBs by a site-specific endonuclease and use them to quantify detected DSBs (labeled, e.g., using i-BLESS). Utilizing qDSB-Seq, we determine numbers of DSBs induced by a radiomimetic drug and replication stress, and reveal two orders of magnitude differences in DSB frequencies. We also measure absolute frequencies of Top1-dependent DSBs at natural replication fork barriers. qDSB-Seq is compatible with various DSB labeling methods in different organisms and allows accurate comparisons of absolute DSB frequencies across samples.


Assuntos
Biologia Computacional/métodos , Quebras de DNA de Cadeia Dupla , Sequenciamento Completo do Genoma/métodos , Linhagem Celular Tumoral , Replicação do DNA/genética , DNA Topoisomerases Tipo I/metabolismo , Genoma Fúngico/genética , Genoma Humano/genética , Humanos , Saccharomycetales/genética
14.
Nat Commun ; 10(1): 2159, 2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31089141

RESUMO

Accurate DNA replication is tightly regulated in eukaryotes to ensure genome stability during cell division and is performed by the multi-protein replisome. At the core an AAA+ hetero-hexameric complex, Mcm2-7, together with GINS and Cdc45 form the active replicative helicase Cdc45/Mcm2-7/GINS (CMG). It is not clear how this replicative ring helicase translocates on, and unwinds, DNA. We measure real-time dynamics of purified recombinant Drosophila melanogaster CMG unwinding DNA with single-molecule magnetic tweezers. Our data demonstrates that CMG exhibits a biased random walk, not the expected unidirectional motion. Through building a kinetic model we find CMG may enter up to three paused states rather than unwinding, and should these be prevented, in vivo fork rates would be recovered in vitro. We propose a mechanism in which CMG couples ATP hydrolysis to unwinding by acting as a lazy Brownian ratchet, thus providing quantitative understanding of the central process in eukaryotic DNA replication.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Drosophila/metabolismo , Modelos Moleculares , DNA Helicases/isolamento & purificação , Proteínas de Drosophila/isolamento & purificação , Fenômenos Magnéticos , Pinças Ópticas , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Imagem Individual de Molécula/métodos
15.
Arch Virol ; 164(8): 2091-2106, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31139938

RESUMO

Beak and feather disease virus (BFDV) belongs to the family Circoviridae. A rolling-circle replication strategy based on a replication-associated protein (Rep) has been proposed for BFDV. The Rep gene of BFDV was expressed and purified, and it was shown to cleave short oligonucleotides containing the conserved nonanucleotide sequence found in the replication origin of circoviruses. This endonuclease activity was most efficient in the presence of the divalent metal ions Mg2+ and Mn2+. Rep proteins containing mutation in the ATPase/GTPase motifs and the 14FTLNN18, 61KKRLS65, 89YCSK92, and 170GKS172 motifs lacked endonuclease activity. The endonuclease activity was not affected by ATPase inhibitors, with the exception of N-ethylmaleimide (NEM), or by GTPase inhibitors, but it was decreased by treatment with the endonuclease inhibitor L-742001. Both the ATPase and GTPase activities were decreased by site-directed mutagenesis and deletion of the ATPase/GTPase and endonuclease motifs. The Rep protein was able to bind a double-stranded DNA fragment of P36 (dsP36) containing the stem-loop structure of the replication origin of BFDV. All of the Rep mutant proteins showed reduced ability to bind this fragment, suggesting that all the ATPase/GTPase and endonuclease motifs are involved in the binding. Other than NEM, all ATPase, GTPase, and endonuclease inhibitors inhibited the binding of the Rep protein to the dsP36 fragment. This is the first report describing the endonuclease activity of the Rep protein of BFDV.


Assuntos
Circovirus/genética , Replicação do DNA/genética , Endonucleases/genética , Replicação Viral/genética , Adenosina Trifosfatases/genética , Infecções por Circoviridae/virologia , DNA Helicases/genética , DNA Viral/genética , GTP Fosfo-Hidrolases/genética , Origem de Replicação/genética , Transativadores/genética
16.
Nat Commun ; 10(1): 2253, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138795

RESUMO

Telomerase negative immortal cancer cells elongate telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. While sustained telomeric replicative stress is required to maintain ALT, it might also lead to cell death when excessive. Here, we show that the ATPase/translocase activity of FANCM keeps telomeric replicative stress in check specifically in ALT cells. When FANCM is depleted in ALT cells, telomeres become dysfunctional, and cells stop proliferating and die. FANCM depletion also increases ALT-associated marks and de novo synthesis of telomeric DNA. Depletion of the BLM helicase reduces the telomeric replication stress and cell proliferation defects induced by FANCM inactivation. Finally, FANCM unwinds telomeric R-loops in vitro and suppresses their accumulation in cells. Overexpression of RNaseH1 completely abolishes the replication stress remaining in cells codepleted for FANCM and BLM. Thus, FANCM allows controlled ALT activity and ALT cell proliferation by limiting the toxicity of uncontrolled BLM and telomeric R-loops.


Assuntos
DNA Helicases/genética , Replicação do DNA/genética , RecQ Helicases/genética , Homeostase do Telômero/genética , Telômero/metabolismo , Morte Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , DNA Helicases/metabolismo , Células HEK293 , Células HeLa , Humanos , RecQ Helicases/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
17.
Nat Commun ; 10(1): 2252, 2019 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-31138797

RESUMO

The collapse of stalled replication forks is a major driver of genomic instability. Several committed mechanisms exist to resolve replication stress. These pathways are particularly pertinent at telomeres. Cancer cells that use Alternative Lengthening of Telomeres (ALT) display heightened levels of telomere-specific replication stress, and co-opt stalled replication forks as substrates for break-induced telomere synthesis. FANCM is a DNA translocase that can form independent functional interactions with the BLM-TOP3A-RMI (BTR) complex and the Fanconi anemia (FA) core complex. Here, we demonstrate that FANCM depletion provokes ALT activity, evident by increased break-induced telomere synthesis, and the induction of ALT biomarkers. FANCM-mediated attenuation of ALT requires its inherent DNA translocase activity and interaction with the BTR complex, but does not require the FA core complex, indicative of FANCM functioning to restrain excessive ALT activity by ameliorating replication stress at telomeres. Synthetic inhibition of FANCM-BTR complex formation is selectively toxic to ALT cancer cells.


Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , RecQ Helicases/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Linhagem Celular Tumoral , Replicação do DNA , Células HCT116 , Células HEK293 , Células HeLa , Humanos
18.
World J Microbiol Biotechnol ; 35(6): 79, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31134410

RESUMO

The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Engenharia Genética/métodos , Pichia/genética , Plasmídeos/genética , Transformação Genética , Replicação do DNA , Escherichia coli/genética , Dosagem de Genes , Regulação Fúngica da Expressão Gênica , Técnicas de Inativação de Genes , Vetores Genéticos , Instabilidade Genômica , Microbiologia Industrial , Kluyveromyces/genética , Regiões Promotoras Genéticas , RNA Guia , Biologia Sintética
19.
Biomed Res Int ; 2019: 3842312, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31058188

RESUMO

There are about 1-2 million follicles presented in the ovary at birth, while only around 1000 primordial follicles are left at menopause. The ovarian function also decreases in parallel with aging. Folliculogenesis is vital for ovarian function, no matter the synthesis of female hormones or ovulation, yet the mechanisms for its changing with increasing age are not fully understood. Early follicle growth up to the large preantral stage is independent of gonadotropins in rodents and relies on intraovarian factors. To further understand the age-related molecular changes in the process of folliculogenesis, we performed microarray gene expression profile analysis using total RNA extracted from young (9 weeks old) and old (32 weeks old) mouse ovarian secondary follicles. The results of our current microarray study revealed that there were 371 (≥2-fold, q-value ≤0.05) genes differentially expressed in which 174 genes were upregulated and 197 genes were downregulated in old mouse ovarian secondary follicles compared to young mouse ovarian secondary follicles. The gene ontology and KEGG pathway analysis of differentially expressed genes uncovered critical biological functions such as immune system process, aging, transcription, DNA replication, DNA repair, protein stabilization, and apoptotic process were affected in the process of aging. The considerable changes in gene expression profile may have an adverse influence on follicle quality and folliculogenesis. Our study provided information on the processes that may contribute to age-related decline in ovarian function.


Assuntos
Envelhecimento/genética , Folículo Ovariano/crescimento & desenvolvimento , Ovário/crescimento & desenvolvimento , RNA/genética , Animais , Reparo do DNA/genética , Replicação do DNA/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Menopausa/genética , Camundongos , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Folículo Ovariano/metabolismo , Ovário/metabolismo , Ovulação/genética , RNA/biossíntese , Transcriptoma/genética
20.
Nat Commun ; 10(1): 2216, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101900

RESUMO

Transcribing and replicating a double-stranded genome require protein modules to unwind, transcribe/replicate nucleic acid substrates, and release products. Here we present in situ cryo-electron microscopy structures of rotavirus dsRNA-dependent RNA polymerase (RdRp) in two states pertaining to transcription. In addition to the previously discovered universal "hand-shaped" polymerase core domain shared by DNA polymerases and telomerases, our results show the function of N- and C-terminal domains of RdRp: the former opens the genome duplex to isolate the template strand; the latter splits the emerging template-transcript hybrid, guides genome reannealing to form a transcription bubble, and opens a capsid shell protein (CSP) to release the transcript. These two "helicase" domains also extensively interact with CSP, which has a switchable N-terminal helix that, like cellular transcriptional factors, either inhibits or promotes RdRp activity. The in situ structures of RdRp, CSP, and RNA in action inform mechanisms of not only transcription, but also replication.


Assuntos
Replicação do DNA/fisiologia , RNA Replicase/ultraestrutura , RNA Mensageiro/ultraestrutura , Rotavirus/fisiologia , Transcrição Genética/fisiologia , Animais , Proteínas do Capsídeo/metabolismo , Proteínas do Capsídeo/ultraestrutura , Linhagem Celular , Cercopithecus aethiops , Microscopia Crioeletrônica , Modelos Moleculares , Domínios Proteicos/genética , RNA Replicase/genética , RNA Replicase/metabolismo , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Rotavirus/ultraestrutura , Replicação Viral/fisiologia
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