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1.
Phys Rev Lett ; 123(3): 038101, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31386470

RESUMO

Synthesis of biopolymers such as DNA, RNA, and proteins are biophysical processes aided by enzymes. The performance of these enzymes is usually characterized in terms of their average error rate and speed. However, because of thermal fluctuations in these single-molecule processes, both error and speed are inherently stochastic quantities. In this Letter, we study fluctuations of error and speed in biopolymer synthesis and show that they are in general correlated. This means that, under equal conditions, polymers that are synthesized faster due to a fluctuation tend to have either better or worse errors than the average. The error-correction mechanism implemented by the enzyme determines which of the two cases holds. For example, discrimination in the forward reaction rates tends to grant smaller errors to polymers with faster synthesis. The opposite occurs for discrimination in monomer rejection rates. Our results provide an experimentally feasible way to identify error-correction mechanisms by measuring the error-speed correlations.


Assuntos
Biopolímeros/biossíntese , Enzimas/química , Enzimas/metabolismo , Biopolímeros/química , DNA/biossíntese , DNA/química , Humanos , Modelos Biológicos , Modelos Químicos , RNA/biossíntese , RNA/química
2.
Yakugaku Zasshi ; 139(7): 969-973, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31257254

RESUMO

Translesion DNA synthesis (TLS) is an emergency system activated to inhibit cell death caused by DNA damage-induced replication arrest. Thus, TLS enables cancer cells to acquire resistance to alkylate anticancer drugs. REV7 functions as the hub protein that interacts with both the inserter DNA polymerase REV1 and the extender DNA polymerase REV3 in TLS. REV7-mediated protein-protein interactions (PPIs) are essential for the activation of TLS, and are therefore attractive targets for anticancer drug development. To clarify the REV7-REV3 and REV7-REV1 PPIs, we determined the structures of REV7-REV3 and REV7-REV3-REV1 complexes. In the structures of REV7-REV3 and REV7-REV3-REV1 complexes, REV7 wraps around the REV3 fragment, and the REV1-binding interface is distinct from the REV3-binding site of REV7. We also identified a novel REV7 binding protein, transcription factor II-I (TFII-I), which is required for TLS. Of note, TFII-I binds the REV7-REV3-REV1 complex, suggesting that REV7-TFII-I PPIs are independent of other REV7-mediated PPIs. Furthermore, we found a small-molecule compound that inhibits TLS by targeting the REV7-REV3 PPIs. Lastly, we determined the structure of REV7 in complex with chromosome alignment maintaining phosphoprotein (CAMP), a known kinetochore-microtubule attachment protein. The overall structure of the REV7-CAMP complex is similar to that of the REV7-REV3 complex, but the REV7-CAMP PPIs are markedly different from the REV7-REV3 PPIs. These findings improve our understanding of multifunctional hub proteins, and are helpful for designing small-molecule compounds for novel anticancer drug development.


Assuntos
Antineoplásicos , Descoberta de Drogas , Proteína Semelhante a ELAV 2/química , Cristalografia por Raios X , DNA/biossíntese , Dano ao DNA , Proteínas de Ligação a DNA/química , DNA Polimerase Dirigida por DNA/química , Humanos , Proteínas Mad2/química , Peso Molecular , Proteínas Nucleares/química , Nucleotidiltransferases/química , Ligação Proteica , Mapas de Interação de Proteínas , Estrutura Terciária de Proteína
3.
Genes Dev ; 33(13-14): 814-827, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171703

RESUMO

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism that occurs in a subset of cancers. One of the hallmarks of ALT cancer is the excessively clustered telomeres in promyelocytic leukemia (PML) bodies, represented as large bright telomere foci. Here, we present a model system that generates telomere clustering in nuclear polySUMO (small ubiquitin-like modification)/polySIM (SUMO-interacting motif) condensates, analogous to PML bodies, and thus artificially engineered ALT-associated PML body (APB)-like condensates in vivo. We observed that the ALT-like phenotypes (i.e., a small fraction of heterogeneous telomere lengths and formation of C circles) are rapidly induced by introducing the APB-like condensates together with BLM through its helicase domain, accompanied by ssDNA generation and RPA accumulation at telomeres. Moreover, these events lead to mitotic DNA synthesis (MiDAS) at telomeres mediated by RAD52 through its highly conserved N-terminal domain. We propose that the clustering of large amounts of telomeres in human cancers promotes ALT that is mediated by MiDAS, analogous to Saccharomyces cerevisiae type II ALT survivors.


Assuntos
Núcleo Celular/metabolismo , DNA/biossíntese , Leucemia Promielocítica Aguda/fisiopatologia , Mitose , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo , Homeostase do Telômero/genética , Motivos de Aminoácidos , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Leucemia Promielocítica Aguda/genética , Fenótipo , Transporte Proteico , Proteína SUMO-1/metabolismo , Telômero/genética , Telômero/metabolismo
4.
Mol Biol (Mosk) ; 53(3): 513-523, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184617

RESUMO

The effects of modified deoxyuridine triphosphates (mod-dUTPs) with different substituents at the C5 position of the pyrimidine cycle on the kinetics of PCR with Taq and Vent (exo-) DNA polymerases are studied. Substituents in mod-dUTP include carboxamide group and groups that are part of the side chains of alanine, valine, leucine, phenylalanine, tryptophan, or tyrosine. For each mod-dUTP, the yields of the target product are measured with the full substitution of dTTP. A fragment of bacterial DNA with a certain nucleotide sequence and a synthetic combinatorial DNA library of random nucleotide sequences are used as templates for amplification. For each mod-dUTP-template-polymerase combination, the correlation between the amplification efficiencies and yields of the target product are investigated. PCR product accumulation curves are influenced by both the template used and the presence of a modified substrate. The catalytic activity of Taq polymerase is higher when mod-dUTPs with short aliphatic substituents are used and decreases when the derivatives with long aliphatic, phenyl, and indole substituents are utilized. Vent (exo-) polymerase is less sensitive to the chemical structure of mod-dUTP. The dynamic measuring of DNA accumulation may be useful for optimizing the temperature-time PCR profiles individually for each of the mod-dUTP. The derivatives may be used in combination with Vent (exo-) polymerase to obtain modified DNA sequences for the method of selection of modified aptamers (mod-SELEX).


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , DNA/química , Reação em Cadeia da Polimerase em Tempo Real , Biblioteca Gênica , Cinética , Técnica de Seleção de Aptâmeros
5.
Methods Mol Biol ; 1973: 1-13, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016692

RESUMO

Chemical modification of nucleic acids can be achieved by the enzymatic polymerization of modified nucleoside triphosphates (dN*TPs). This approach obviates some of the requirements and drawbacks imposed by the more traditional solid-phase synthesis of oligonucleotides. Here, we describe the protocol that is necessary to synthesize dN*TPs and evaluate their substrate acceptance by polymerases for their subsequent use in various applications including selection experiments to identify aptamers. The protocol is exemplified for a sugar-constrained nucleoside analog, 7',5'-bc-TTP.


Assuntos
Compostos Bicíclicos com Pontes/química , DNA/biossíntese , Nucleotídeos/química , Açúcares/química , DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Oligonucleotídeos/química , Técnicas de Síntese em Fase Sólida
6.
Methods Mol Biol ; 1973: 15-25, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016693

RESUMO

Formation of adducts to DNA is of great benefit to DNA sequencing and damage detection technology and to enzymology. Here we describe the synthesis and characterization procedures of 18-crown-6 adducts formed to abasic (AP) sites, 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG), and 2'-deoxycytidine (C) residues in DNA oligodeoxynucleotides. These crown ether adducts were used as site-specific modifications to facilitate nanopore technology. The methods described can be readily expanded to attach other suitable primary amines of interest.


Assuntos
Éteres de Coroa/química , Adutos de DNA/química , DNA/biossíntese , Desoxicitidina/química , Desoxiguanosina/análogos & derivados , Oligodesoxirribonucleotídeos/química , DNA/química , Desoxiguanosina/química , Nanoporos
7.
Methods Mol Biol ; 1973: 39-57, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016695

RESUMO

Synthesis of base-modified dNTPs through the Suzuki or Sonogashira cross-coupling reactions of halogenated dNTPs with boronic acids or alkynes is reported, as well as the use of these modified dNTPs in polymerase incorporations to oligonucleotides or DNA by primer extension or PCR.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/biossíntese , Nucleotídeos/biossíntese , DNA/química , Halogenação , Nucleotídeos/química
8.
Methods Mol Biol ; 1973: 59-89, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016696

RESUMO

This chapter describes procedures for (1) the synthesis of six 2'-C,4'-C-ethyleneoxy-bridged thymidine phosphoramidites, i.e., methylene-EoDNA-T, (R)-Me-methylene-EoDNA-T, (S)-Me-methylene-EoDNA-T, EoDNA-T, (R)-Me-EoDNA-T, and (S)-Me-EoDNA-T phosphoramidites, (2) the introduction of the phosphoramidites into oligonucleotides, (3) UV-melting experiments of the duplexes of the modified oligonucleotides and complementary RNA, and (4) nuclease degradation experiments of the modified oligonucleotides.


Assuntos
Compostos Bicíclicos com Pontes/química , DNA/biossíntese , DNA/química , Desoxirribonucleases/metabolismo , Etilenos/química , Timina/química , Estabilidade Enzimática
9.
Gen Comp Endocrinol ; 276: 69-76, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30851298

RESUMO

The function of oocyte-derived growth differentiation factor 9 (GDF9) in ovarian follicles has thus far been poorly defined in avian species compared with the defined function in mammals. Our aim here is to investigate the effects of GDF9 on steroidogenesis and on chicken ovarian granulosa cell (GC) mitosis. Primary GCs from both prehierarchical (6-8 mm in diameter, phGCs) and preovulatory follicles (F1-F5, poGCs) were cultured in the presence or absence of the GDF9 protein. The progesterone (P4) levels in the culture medium were then measured by radioimmunoassay (RIA), and the expression levels of steroidogenesis genes were detected by quantitative PCR. We found that GDF9 alone showed no significant effect on the P4 levels by regulating the expression of steroidogenesis genes, such as STAR, CYP11A1 and HSD3B. Further experiments indicated that GDF9 promoted follicle-stimulating hormone (FSH)-induced P4 production and STAR expression. GDF9 also rescued the FSH-induced decrease of FSH receptor (FSHR) expression but had no effect on the forskolin-induced P4, STAR and forskolin-inhibited FSHR expression levels, suggesting that GDF9 might achieve its regulatory role of P4 by enhancing FSHR and STAR expression. In addition, GDF9 also promoted GC cell cycle progression, regulated the gene transcription of related genes, potentiated DNA replication and inhibited apoptosis. Interestingly, these effects differed between the phGCs and the poGCs. To our knowledge, this is the first report that illustrates the function of GDF9 on chicken GCs and the effects on ovarian steroidogenesis. Our findings highlight the regulation of central oocytes on the surrounding granulosa cells and emphasize the interaction between paracrine signals and endocrine hormones on ovarian progesterone production; these findings contribute to the understanding of the development of avian ovarian follicles.


Assuntos
Galinhas/metabolismo , Hormônio Foliculoestimulante/farmacologia , Células da Granulosa/metabolismo , Fator 9 de Diferenciação de Crescimento/farmacologia , Progesterona/biossíntese , Animais , Apoptose/efeitos dos fármacos , Proteínas Aviárias/genética , Proteínas Aviárias/metabolismo , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Colforsina/farmacologia , DNA/biossíntese , Replicação do DNA/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/efeitos dos fármacos , Humanos , Ovulação/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Radioimunoensaio
10.
Mol Cell ; 73(5): 915-929.e6, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30849395

RESUMO

DNA replication errors generate complex chromosomal rearrangements and thereby contribute to tumorigenesis and other human diseases. One mechanism that triggers these errors is mitotic entry before the completion of DNA replication. To address how mitosis might affect DNA replication, we used Xenopus egg extracts. When mitotic CDK (Cyclin B1-CDK1) is used to drive interphase egg extracts into a mitotic state, the replicative CMG (CDC45/MCM2-7/GINS) helicase undergoes ubiquitylation on its MCM7 subunit, dependent on the E3 ubiquitin ligase TRAIP. Whether replisomes have stalled or undergone termination, CMG ubiquitylation is followed by its extraction from chromatin by the CDC48/p97 ATPase. TRAIP-dependent CMG unloading during mitosis is also seen in C. elegans early embryos. At stalled forks, CMG removal results in fork breakage and end joining events involving deletions and templated insertions. Our results identify a mitotic pathway of global replisome disassembly that can trigger replication fork collapse and DNA rearrangements.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclina B1/metabolismo , Dano ao DNA , Replicação do DNA , DNA/biossíntese , Rearranjo Gênico , Mitose , Proteínas Quinases/metabolismo , Proteínas de Xenopus/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/genética , Ciclina B1/genética , DNA/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo
11.
Nat Commun ; 10(1): 1185, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30862852

RESUMO

Cellular products derived from the activity of DNA, RNA, and protein synthesis collectively control cell identity and function. Yet there is little information on how these three biosynthesis activities are coordinated during transient and sparse cellular processes, such as activation and differentiation. Here, we describe Simultaneous Overview of tri-Molecule Biosynthesis (SOM3B), a molecular labeling and simultaneous detection strategy to quantify DNA, RNA, and protein synthesis in individual cells. Comprehensive interrogation of biosynthesis activities during transient cell states, such as progression through cell cycle or cellular differentiation, is achieved by partnering SOM3B with parallel quantification of select biomolecules with conjugated antibody reagents. Here, we investigate differential de novo DNA, RNA, and protein synthesis dynamics in transformed human cell lines, primary activated human immune cells, and across the healthy human hematopoietic continuum, all at a single-cell resolution.


Assuntos
DNA/biossíntese , Biossíntese de Proteínas , RNA/biossíntese , Análise de Célula Única/métodos , Medula Óssea/metabolismo , Ciclo Celular , Células HEK293 , Células HeLa , Voluntários Saudáveis , Humanos , Células Jurkat , Leucócitos Mononucleares , Cultura Primária de Células , Coloração e Rotulagem/métodos
12.
Nature ; 567(7747): 267-272, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30842657

RESUMO

Cells often use multiple pathways to repair the same DNA lesion, and the choice of pathway has substantial implications for the fidelity of genome maintenance. DNA interstrand crosslinks covalently link the two strands of DNA, and thereby block replication and transcription; the cytotoxicity of these crosslinks is exploited for chemotherapy. In Xenopus egg extracts, the collision of replication forks with interstrand crosslinks initiates two distinct repair pathways. NEIL3 glycosylase can cleave the crosslink1; however, if this fails, Fanconi anaemia proteins incise the phosphodiester backbone that surrounds the interstrand crosslink, generating a double-strand-break intermediate that is repaired by homologous recombination2. It is not known how the simpler NEIL3 pathway is prioritized over the Fanconi anaemia pathway, which can cause genomic rearrangements. Here we show that the E3 ubiquitin ligase TRAIP is required for both pathways. When two replisomes converge at an interstrand crosslink, TRAIP ubiquitylates the replicative DNA helicase CMG (the complex of CDC45, MCM2-7 and GINS). Short ubiquitin chains recruit NEIL3 through direct binding, whereas longer chains are required for the unloading of CMG by the p97 ATPase, which enables the Fanconi anaemia pathway. Thus, TRAIP controls the choice between the two known pathways of replication-coupled interstrand-crosslink repair. These results, together with our other recent findings3,4 establish TRAIP as a master regulator of CMG unloading and the response of the replisome to obstacles.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , DNA/química , DNA/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , DNA/biossíntese , Replicação do DNA , Feminino , Humanos , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , N-Glicosil Hidrolases/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitinação , Xenopus
14.
Proc Natl Acad Sci U S A ; 116(8): 3042-3051, 2019 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-30718400

RESUMO

Transcription termination is a critical step in the control of gene expression. One of the major termination mechanisms is mediated by Rho factor that dissociates the complex mRNA-DNA-RNA polymerase upon binding with RNA polymerase. Rho promotes termination at the end of operons, but it can also terminate transcription within leader regions, performing regulatory functions and avoiding pervasive transcription. Transcription of rho is autoregulated through a Rho-dependent attenuation in the leader region of the transcript. In this study, we have included an additional player in this pathway. By performing MS2-affinity purification coupled with RNA sequencing (MAPS), rho transcript was shown to directly interact with the small noncoding RNA SraL. Using bioinformatic in vivo and in vitro experimental analyses, SraL was shown to base pair with the 5'-UTR of rho mRNA upregulating its expression in several growth conditions. This base pairing was shown to prevent the action of Rho over its own message. Moreover, the results obtained indicate that both ProQ and Hfq are associated with this regulation. We propose a model that contemplates the action of Salmonella SraL sRNA in the protection of rho mRNA from premature transcription termination by Rho. Note that since the interaction region between both RNAs corresponds to a very-well-conserved sequence, it is plausible to admit that this regulation also occurs in other enterobacteria.


Assuntos
DNA/genética , Pequeno RNA não Traduzido/genética , Fator Rho/genética , Terminação da Transcrição Genética , DNA/biossíntese , RNA Polimerases Dirigidas por DNA/genética , Regulação Bacteriana da Expressão Gênica/genética , Conformação de Ácido Nucleico , RNA Mensageiro/genética , Salmonella enterica/genética , Análise de Sequência de RNA , Transcrição Genética
15.
J Biochem ; 165(6): 505-516, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30649446

RESUMO

The Cullin-RING ubiquitin ligase CRL4Cdt2 maintains genome integrity by mediating the cell cycle- and DNA damage-dependent degradation of proteins such as Cdt1, p21 and Set8. Human Cdt2 has two regions, a conserved N-terminal seven WD40 repeat region and a less conserved C-terminal region. Here, we showed that the N-terminal region is sufficient for complex formation with CRL4, but the C-terminal region is required for the full ubiquitin ligase activity. UV irradiation-induced polyubiquitination and degradation of Cdt1 were impaired in Cdt2 (N-terminus only)-expressing cells. Deletion and mutation analysis identified a domain in the C-terminal region that increased ubiquitination activity and displayed DNA-binding activity. The identified domain mediated binding to double-stranded DNA and showed higher affinity binding to single-stranded DNA. As the ligase activity of CRL4Cdt2 depends on proliferating cell nuclear antigen (PCNA) loading onto DNA, the present results suggest that the DNA-binding domain facilitates the CRL4Cdt2-mediated recognition and ubiquitination of substrates bound to PCNA on chromatin.


Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA/biossíntese , Proteínas Nucleares/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sítios de Ligação , Células Cultivadas , DNA/química , Humanos
16.
Mol Cell ; 73(3): 574-588.e7, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30595436

RESUMO

DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.


Assuntos
Reparo do DNA , Replicação do DNA , DNA/biossíntese , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animais , DNA/química , DNA/genética , Feminino , Masculino , Conformação de Ácido Nucleico , Complexo de Endopeptidases do Proteassoma/genética , Domínios e Motivos de Interação entre Proteínas , Proteólise , Células Sf9 , Relação Estrutura-Atividade , Ubiquitinação , Proteínas de Xenopus/genética , Xenopus laevis/genética
17.
J Dairy Sci ; 102(3): 2618-2630, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30612800

RESUMO

The objective of this study was to analyze the mammary gland transcriptome to determine how preweaning nutrient supply alters the molecular mechanisms that regulate preweaning mammary development. Holstein heifers were fed via milk replacer (MR) either an elevated level of nutrient intake (ELE; on average, 5.9 ± 0.2 Mcal of ME in 8.4 L of MR/d, n = 6) or a restricted amount of nutrients (RES; 2.8 ± 0.2 Mcal of ME in 4 L of MR/d, n = 5) for 54 d after birth, at which point they were slaughtered and samples of mammary parenchyma tissue were obtained. Parenchymal mRNA was analyzed, and the fold change (FC) of 18,111 genes (ELE relative to RES) was uploaded to Ingenuity Pathway Analysis (IPA) software (Qiagen Bioinformatics, Redwood City, CA) for transcriptomic analysis. Using a threshold of P < 0.05, IPA identified that the FC of 1,931 of 18,811 differentially expressed genes (DEG) could be used for the analysis. A total of 18 molecular and cellular functions were relevant to DEG arising from the treatments; the 5 functions most associated with DEG were cell death and survival, cellular movement, cellular development, cellular growth and proliferation, and lipid metabolism. Based on the directional FC of DEG, the mammary gland of ELE heifers was predicted to have increased epithelial-mesenchymal transition (Z = 2.685) and accumulation of lipid (Z = 2.322), whereas the synthesis of DNA (Z = -2.137), transactivation of RNA (Z = -2.254), expression of RNA (Z = -2.405), transcription (Z = -2.482), and transactivation (Z = -2.611) were all predicted to be decreased. Additionally, IPA predicted the activation status of 13 upstream regulators with direct influence on DEG as affected by ELE feeding that were ligand-dependent nuclear receptors (n = 2), enzymes (n = 1), or transcription regulators (n = 10). Of these, 6 were activated (Z > 2) and 7 were inhibited (Z < -2). In summary, feeding ELE preweaning altered the mammary transcriptome of Holstein heifers, affecting cell functions involved in the morphological and physiological development of the mammary gland.


Assuntos
Bovinos/metabolismo , Perfilação da Expressão Gênica/veterinária , Regulação da Expressão Gênica/fisiologia , Glândulas Mamárias Animais/metabolismo , Nutrientes/administração & dosagem , Desmame , Ração Animal/análise , Animais , Proliferação de Células , DNA/biossíntese , Dieta/veterinária , Ingestão de Energia , Feminino , Metabolismo dos Lipídeos/fisiologia , Leite , RNA/genética , RNA Mensageiro/análise
18.
Nucleic Acids Res ; 47(4): 1836-1846, 2019 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-30544167

RESUMO

Bacterial recombinational repair of double-strand breaks often begins with creation of initiating 3' single-stranded DNA (ssDNA) tails on each side of a double-strand break (DSB). Importantly, if the RecBCD pathway is followed, RecBCD creates a gap between the sequences at 3' ends of the initiating strands. The gap flanks the DSB and extends at least to the nearest Chi site on each strand. Once the initiating strands form ssDNA-RecA filaments, each ssDNA-RecA filament searches for homologous double-stranded DNA (dsDNA) to use as a template for the DNA synthesis needed to fill the gap created by RecBCD. Our experimental results show that the DNA synthesis requires formation of a heteroduplex dsDNA that pairs >20 contiguous bases in the initiating strand with sequence matched bases in a strand from the original dsDNA. To trigger synthesis, the heteroduplex must be near the 3' end of the initiating strand. Those experimentally determined requirements for synthesis combined with the Chi site dependence of the function of RecBCD and the distribution of Chi sites in bacterial genomes could allow the RecBCD pathway to avoid some genomic rearrangements arising from directly induced DSBs; however, the same three factors could promote other rearrangements.


Assuntos
DNA/genética , Exodesoxirribonuclease V/genética , Genoma Bacteriano/genética , Sequência de Bases , DNA/biossíntese , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Replicação do DNA/genética , DNA de Cadeia Simples/genética , Escherichia coli/genética , Ácidos Nucleicos Heteroduplexes/genética , Recombinases Rec A/genética , Recombinação Genética
19.
PLoS One ; 13(12): e0208947, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30532129

RESUMO

Purines are essential molecules for all forms of life. In addition to constituting a backbone of DNA and RNA, purines play roles in many metabolic pathways, such as energy utilization, regulation of enzyme activity, and cell signaling. The supply of purines is provided by two pathways: the salvage pathway and de novo synthesis. Although purine de novo synthesis (PDNS) activity varies during the cell cycle, this pathway represents an important source of purines, especially for rapidly dividing cells. A method for the detailed study of PDNS is lacking for analytical reasons (sensitivity) and because of the commercial unavailability of the compounds. The aim was to fully describe the mass spectrometric fragmentation behavior of newly synthesized PDNS-related metabolites and develop an analytical method. Except for four initial ribotide PDNS intermediates that preferentially lost water or phosphate or cleaved the forming base of the purine ring, all the other metabolites studied cleaved the glycosidic bond in the first fragmentation stage. Fragmentation was possible in the third to sixth stages. A liquid chromatography-high-resolution mass spectrometric method was developed and applied in the analysis of CRISPR-Cas9 genome-edited HeLa cells deficient in the individual enzymatic steps of PDNS and the salvage pathway. The identities of the newly synthesized intermediates of PDNS were confirmed by comparing the fragmentation patterns of the synthesized metabolites with those produced by cells (formed under pathological conditions of known and theoretically possible defects of PDNS). The use of stable isotope incorporation allowed the confirmation of fragmentation mechanisms and provided data for future fluxomic experiments. This method may find uses in the diagnosis of PDNS disorders, the investigation of purinosome formation, cancer research, enzyme inhibition studies, and other applications.


Assuntos
DNA/biossíntese , Purinas/biossíntese , RNA/biossíntese , Espectrometria de Massas em Tandem , Sistemas CRISPR-Cas , Cromatografia Líquida , DNA/química , Edição de Genes , Células HeLa , Humanos , Purinas/química , RNA/química
20.
Biomed Res Int ; 2018: 1736738, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30539004

RESUMO

Autophagy plays an important role in tumor development because of its capacity to maintain energy homeostasis by recycling damaged intracellular proteins and organelles, and increased autophagy levels are reported to mediate drug resistance in many cancers. However, whether high autophagy levels negatively impact tumor cell growth is unknown. Herein, we found that cisplatin (ddp)-resistant cells were more sensitive to glutamine (Gln) deprivation than ddp-sensitive cells, and they showed significant G1 arrest and increased apoptosis rates under Gln-deficient conditions. Furthermore, ddp-resistant cells had a higher level of autophagy, which mediated ddp resistance. Further analysis indicated that Gln deficiency could trigger apoptosis by enhancing activation of the autophagy signaling pathway AMPK/ULK1 in ddp-resistant cells due to their high basal autophagy level. Interestingly, ddp-resistant cells were more sensitive to rapamycin, and rapamycin could efficiently suppress the growth of ddp-resistant cells in vivo. Taken together, our study demonstrated that ddp-resistant cells became vulnerable to Gln deprivation because of their increased level of autophagy, and for the first time, we showed that suppressing the growth of ddp-resistant cells via enhancing autophagy induction was possible with rapamycin treatment.


Assuntos
Autofagia/efeitos dos fármacos , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , DNA/biossíntese , Glutamina/metabolismo , Humanos , Masculino , Camundongos Nus , Sirolimo/farmacologia
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