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1.
Cell Rep ; 28(10): 2673-2688.e8, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31484077

RESUMO

In the eukaryotic replisome, DNA unwinding by the Cdc45-MCM-Go-Ichi-Ni-San (GINS) (CMG) helicase requires a hexameric ring-shaped ATPase named minichromosome maintenance (MCM), which spools single-stranded DNA through its central channel. Not all six ATPase sites are required for unwinding; however, the helicase mechanism is unknown. We imaged ATP-hydrolysis-driven translocation of the CMG using cryo-electron microscopy (cryo-EM) and found that the six MCM subunits engage DNA using four neighboring protomers at a time, with ATP binding promoting DNA engagement. Morphing between different helicase states leads us to suggest a non-symmetric hand-over-hand rotary mechanism, explaining the asymmetric requirements of ATPase function around the MCM ring of the CMG. By imaging of a higher-order replisome assembly, we find that the Mrc1-Csm3-Tof1 fork-stabilization complex strengthens the interaction between parental duplex DNA and the CMG at the fork, which might support the coupling between DNA translocation and fork unwinding.


Assuntos
Trifosfato de Adenosina/metabolismo , DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Eucariotos/enzimologia , Complexos Multienzimáticos/metabolismo , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Animais , Microscopia Crioeletrônica , DNA/ultraestrutura , DNA Helicases/química , DNA Helicases/ultraestrutura , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/enzimologia , Hidrólise , Modelos Moleculares , Domínios Proteicos , Saccharomyces cerevisiae/metabolismo
2.
Sci Rep ; 8(1): 12136, 2018 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-30108253

RESUMO

Coordination of DNA replication and cellular redox homeostasis mechanisms is essential for the sustained genome stability due to the sensitivity of replicating DNA to oxidation. However, substantial gaps remain in our knowledge of underlying molecular pathways. In this study, we characterise the interaction of Keap1, a central antioxidant response regulator in Metazoa, with the replicative helicase subunit protein MCM3. Our analysis suggests that structural determinants of the interaction of Keap1 with its critical downstream target - Nrf2 master transactivator of oxidative stress response genes - may have evolved in evolution to mimic the conserved helix-2-insert motif of MCM3. We show that this has led to a competition between MCM3 and Nrf2 proteins for Keap1 binding, and likely recruited MCM3 for the competitive binding dependent modulation of Keap1 controlled Nrf2 activities. We hypothesise that such mechanism could help to adjust the Keap1-Nrf2 antioxidant response pathway according to the proliferative and replicative status of the cell, with possible reciprocal implications also for the regulation of cellular functions of MCM3. Altogether this suggests about important role of Keap1-MCM3 interaction in the cross-talk between replisome and redox homeostasis machineries in metazoan cells.


Assuntos
Replicação do DNA , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Componente 3 do Complexo de Manutenção de Minicromossomo/metabolismo , Estresse Oxidativo/fisiologia , Motivos de Aminoácidos , Animais , Células CHO , Linhagem Celular Tumoral , Cricetulus , Evolução Molecular , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/química , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/isolamento & purificação , Queratinócitos , Componente 3 do Complexo de Manutenção de Minicromossomo/química , Componente 3 do Complexo de Manutenção de Minicromossomo/genética , Componente 3 do Complexo de Manutenção de Minicromossomo/isolamento & purificação , Fator 2 Relacionado a NF-E2/metabolismo , Cultura Primária de Células , Ligação Proteica/fisiologia , Conformação Proteica em alfa-Hélice , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Células Sf9 , Spodoptera , Transativadores/metabolismo
3.
J Virol ; 89(21): 11030-45, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26311875

RESUMO

UNLABELLED: The A7(74) strain of Semliki Forest virus (SFV; genus Alphavirus) is avirulent in adult mice, while the L10 strain is virulent in mice of all ages. It has been previously demonstrated that this phenotypic difference is associated with nonstructural protein 3 (nsP3). Consensus clones of L10 (designated SFV6) and A7(74) (designated A774wt) were used to construct a panel of recombinant viruses. The insertion of nsP3 from A774wt into the SFV6 backbone had a minor effect on the virulence of the resulting recombinant virus. Conversely, insertion of nsP3 from SFV6 into the A774wt backbone or replacement of A774wt nsP3 with two copies of nsP3 from SFV6 resulted in virulent viruses. Unexpectedly, duplication of nsP3-encoding sequences also resulted in elevated levels of nsP4, revealing that nsP3 is involved in the stabilization of nsP4. Interestingly, replacement of nsP3 of SFV6 with that of A774wt resulted in a virulent virus; the virulence of this recombinant was strongly reduced by functionally coupled substitutions for amino acid residues 534 (P4 position of the cleavage site between nsP1 and nsP2) and 1052 (S4 subsite residue of nsP2 protease) in the nonstructural polyprotein. Pulse-chase experiments revealed that A774wt and avirulent recombinant virus were characterized by increased processing speed of the cleavage site between nsP1 and nsP2. A His534-to-Arg substitution specifically activated this cleavage, while a Val1052-to-Glu substitution compensated for this effect by reducing the basal protease activity of nsP2. These findings provide a link between nonstructural polyprotein processing and the virulence of SFV. IMPORTANCE: SFV infection of mice provides a well-characterized model to study viral encephalitis. SFV also serves as a model for studies of alphavirus molecular biology and host-pathogen interactions. Thus far, the genetic basis of different properties of SFV strains has been studied using molecular clones, which often contain mistakes originating from standard cDNA synthesis and cloning procedures. Here, for the first time, consensus clones of SFV strains were used to map virulence determinants. Existing data on the importance of nsP3 for virulent phenotypes were confirmed, another determinant of neurovirulence and its molecular basis was characterized, and a novel function of nsP3 was identified. These findings provide links between the molecular biology of SFV and its biological properties and significantly increase our understanding of the basis of alphavirus-induced pathology. In addition, the usefulness of consensus clones as tools for studies of alphaviruses was demonstrated.


Assuntos
Neurônios/virologia , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes/metabolismo , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/patogenicidade , Proteínas não Estruturais Virais/genética , Substituição de Aminoácidos/genética , Animais , Linhagem Celular , DNA Complementar/biossíntese , Immunoblotting , Camundongos , Microscopia de Fluorescência , Processamento de Proteína Pós-Traducional/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Vírus da Floresta de Semliki/metabolismo , Estatísticas não Paramétricas , Virulência
4.
J Gen Virol ; 88(Pt 4): 1225-1230, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-17374766

RESUMO

Alphavirus-based vector and replicon systems have been extensively used experimentally and are likely to be used in human and animal medicine. Whilst marker genes can be inserted easily under the control of a duplicated subgenomic promoter, these constructs are often genetically unstable. Here, a novel alphavirus construct is described in which an enhanced green fluorescent protein (EGFP) marker gene is inserted into the virus replicase open reading frame between nsP3 and nsP4, flanked by nsP2 protease-recognition sites. This construct has correct processing of the replicase polyprotein, produces viable virus and expresses detectable EGFP fluorescence upon infection of cultured cells and cells of the mouse brain. In comparison to parental virus, the marker virus has an approximately 1 h delay in virus RNA and infectious virus production. Passage of the marker virus in vitro and in vivo demonstrates good genetic stability. Insertion of different markers into this novel construct has potential for various applications.


Assuntos
Proteínas de Fluorescência Verde/biossíntese , RNA Polimerase Dependente de RNA/genética , Vírus da Floresta de Semliki/genética , Proteínas não Estruturais Virais/genética , Animais , Encéfalo/virologia , Linhagem Celular , Cricetinae , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Camundongos , RNA Polimerase Dependente de RNA/fisiologia , Vírus da Floresta de Semliki/crescimento & desenvolvimento , Coloração e Rotulagem , Proteínas não Estruturais Virais/fisiologia , Replicação Viral
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