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1.
Proc Natl Acad Sci U S A ; 114(4): E466-E475, 2017 01 24.
Artigo em Inglês | MEDLINE | ID: mdl-28069956

RESUMO

Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show that Escherichia coli RecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure. Through single-molecule and ensemble biophysical experiments, we elucidate how the conserved domain architecture of RecQ supports geometry-dependent shuttling and directed processing of recombination-intermediate [displacement loop (D-loop)] substrates. Our study shows that these activities together suppress illegitimate recombination in vivo, whereas unregulated duplex unwinding is detrimental for recombination precision. Based on these results, we propose a mechanism through which RecQ helicases achieve recombination precision and efficiency.


Assuntos
DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Recombinação Homóloga , RecQ Helicases/metabolismo , Reparo do DNA , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/química , Sequências Repetidas Invertidas , RecQ Helicases/química
2.
Nucleic Acids Res ; 45(20): 11878-11890, 2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29059328

RESUMO

The single-stranded DNA binding protein (SSB) of Escherichia coli plays essential roles in maintaining genome integrity by sequestering ssDNA and mediating DNA processing pathways through interactions with DNA-processing enzymes. Despite its DNA-sequestering properties, SSB stimulates the DNA processing activities of some of its binding partners. One example is the genome maintenance protein RecQ helicase. Here, we determine the mechanistic details of the RecQ-SSB interaction using single-molecule magnetic tweezers and rapid kinetic experiments. Our results reveal that the SSB-RecQ interaction changes the binding mode of SSB, thereby allowing RecQ to gain access to ssDNA and facilitating DNA unwinding. Conversely, the interaction of RecQ with the SSB C-terminal tail increases the on-rate of RecQ-DNA binding and has a modest stimulatory effect on the unwinding rate of RecQ. We propose that this bidirectional communication promotes efficient DNA processing and explains how SSB stimulates rather than inhibits RecQ activity.


Assuntos
DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , RecQ Helicases/metabolismo , DNA Bacteriano/química , DNA Bacteriano/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Magnetismo , Modelos Moleculares , Conformação de Ácido Nucleico , Pinças Ópticas , Ligação Proteica , Domínios Proteicos , RecQ Helicases/química
3.
J Biol Chem ; 289(9): 5938-49, 2014 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-24403069

RESUMO

The allosteric communication between the ATP- and DNA-binding sites of RecQ helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded DNA (ssDNA) and, in turn, the restructuring of multistranded DNA substrates during genome maintenance processes. In this study, we used the tryptophan fluorescence signal of Escherichia coli RecQ helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. Rapid kinetic experiments revealed that ssDNA binding occurs in a two-step mechanism in which the initial binding step is followed by a structural transition of the DNA-bound helicase. We found that the nucleotide state of RecQ greatly influences the kinetics of the detected structural transition, which leads to a high affinity DNA-clamped state in the presence of the nucleotide analog ADP-AlF4. The DNA binding mechanism is largely independent of ssDNA length, indicating the independent binding of RecQ molecules to ssDNA and the lack of significant DNA end effects. The structural transition of DNA-bound RecQ was not detected when the ssDNA binding capability of the helicase-RNase D C-terminal domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multistranded DNA processing by RecQ helicases.


Assuntos
DNA Bacteriano/química , DNA de Cadeia Simples/química , Escherichia coli/enzimologia , RecQ Helicases/química , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Cinética , Compostos Organometálicos/química , Estrutura Terciária de Proteína , RecQ Helicases/genética , RecQ Helicases/metabolismo , Ribonuclease III/química
4.
Viruses ; 12(1)2020 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-31968613

RESUMO

The West Nile virus is endemic in multiple European countries and responsible for several epidemics throughout the European region. Its evolution into local or even widespread epidemics is driven by multiple factors from genetic diversification of the virus to environmental conditions. The year of 2018 was characterized by an extraordinary increase in human and animal cases in the Central-Eastern European region, including Hungary. In a collaborative effort, we summarized and analyzed the genetic and serologic data of WNV infections from multiple Hungarian public health institutions, universities, and private organizations. We compared human and veterinary serologic data, along with NS5 and NS3 gene sequence data through 2018. Wild birds were excellent indicator species for WNV circulation in each year. Our efforts resulted in documenting the presence of multiple phylogenetic subclades with Balkans and Western-European progenitor sequences of WNV circulating among human and animal populations in Hungary prior to and during the 2018 epidemic. Supported by our sequence and phylogenetic data, the epidemic of 2018 was not caused by recently introduced WNV strains. Unfortunately, Hungary has no country-wide integrated surveillance system which would enable the analysis of related conditions and provide a comprehensive epidemiological picture. The One Health approach, involving multiple institutions and experts, should be implemented in order to fully understand ecological background factors driving the evolution of future epidemics.


Assuntos
Cavalos/virologia , Filogenia , Proteínas Virais , Vírus do Nilo Ocidental , Animais , Antígenos Virais/genética , Antígenos Virais/imunologia , Aves/virologia , Encefalite/virologia , Epidemias , Genes Virais , Falcões/virologia , Humanos , Hungria/epidemiologia , Saúde Única , Patologia Molecular , Estudos Soroepidemiológicos , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Febre do Nilo Ocidental/veterinária , Vírus do Nilo Ocidental/genética , Vírus do Nilo Ocidental/imunologia , Vírus do Nilo Ocidental/isolamento & purificação
5.
Sci Rep ; 5: 11091, 2015 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-26067769

RESUMO

DNA-restructuring activities of RecQ-family helicases play key roles in genome maintenance. These activities, driven by two tandem RecA-like core domains, are thought to be controlled by accessory DNA-binding elements including the helicase-and-RnaseD-C-terminal (HRDC) domain. The HRDC domain of human Bloom's syndrome (BLM) helicase was shown to interact with the RecA core, raising the possibility that it may affect the coupling between ATP hydrolysis, translocation along single-stranded (ss)DNA and/or unwinding of double-stranded (ds)DNA. Here, we determined how these activities are affected by the abolition of the ssDNA interaction of the HRDC domain or the deletion of the entire domain in E. coli RecQ helicase. Our data show that the HRDC domain suppresses the rate of DNA-activated ATPase activity in parallel with those of ssDNA translocation and dsDNA unwinding, regardless of the ssDNA binding capability of this domain. The HRDC domain does not affect either the processivity of ssDNA translocation or the tight coupling between the ATPase, translocation, and unwinding activities. Thus, the mechanochemical coupling of E. coli RecQ appears to be independent of HRDC-ssDNA and HRDC-RecA core interactions, which may play roles in more specialized functions of the enzyme.


Assuntos
DNA Bacteriano/química , DNA de Cadeia Simples/química , Escherichia coli/enzimologia , RecQ Helicases/química , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Estabilidade Enzimática , Humanos , Estrutura Terciária de Proteína , Recombinases Rec A/química , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo
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