RESUMO
Minichromosome maintenance 2-7 (MCM2-7) proteins are conserved eukaryotic replicative factors essential for the DNA replication at its initiation and elongation step, and act as a licensing factor. The MCM2-7 and MCM4/6/7subcomplex exhibit DNA helicase activity, and are therefore regarded as the replicative helicase. The MCM proteins have not been studied in detail in plant system. Here, we present the biochemical characterization of Arabidopsis thaliana MCM3 single subunit and show that it exhibits in vitro unwinding and ATPase activities. AtMCM3 shows a greater unwinding activity with 5' forked partial DNA duplex substrate as compared to 3' forked and non-forked substrates. ATP and magnesium ion are indispensable for its DNA helicase activity. Specifically, ATP and dATP are the preferred nucleotides for its unwinding activity. The directionality of the AtMCM3 has been determined to be in 3' to 5' direction. The oligomerization status of AtMCM3 single subunit protein indicates that it is present in different multimeric forms. The unraveling of the helicase activity of AtMCM3 will provide better insights into the plant DNA replication.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Componente 3 do Complexo de Manutenção de Minicromossomo/química , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Proteínas de Arabidopsis/fisiologia , Biocatálise , DNA de Plantas/química , DNA de Cadeia Simples/química , Hidrólise , Componente 3 do Complexo de Manutenção de Minicromossomo/fisiologia , Ligação Proteica , Estrutura Quaternária de ProteínaRESUMO
The nonstructural protein NSs, encoded by the S RNA of groundnut bud necrosis virus (GBNV) (genus Tospovirus, family Bunyaviridae) has earlier been shown to possess nucleic-acid-stimulated NTPase and 5' α phosphatase activity. ATP hydrolysis is an essential function of a true helicase. Therefore, NSs was tested for DNA helicase activity. The results demonstrated that GBNV NSs possesses bidirectional DNA helicase activity. An alanine mutation in the Walker A motif (K189A rNSs) decreased DNA helicase activity substantially, whereas a mutation in the Walker B motif resulted in a marginal decrease in this activity. The parallel loss of the helicase and ATPase activity in the K189A mutant confirms that NSs acts as a non-canonical DNA helicase. Furthermore, both the wild-type and K189A NSs could function as RNA silencing suppressors, demonstrating that the suppressor activity of NSs is independent of its helicase or ATPase activity. This is the first report of a true helicase from a negative-sense RNA virus.
Assuntos
DNA Helicases/metabolismo , Tospovirus/enzimologia , Proteínas Virais/metabolismo , Motivos de Aminoácidos , DNA Helicases/química , DNA Helicases/genética , DNA Viral/genética , Inativação Gênica , Mutação de Sentido Incorreto , Doenças das Plantas/genética , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Nicotiana/genética , Nicotiana/virologia , Tospovirus/química , Tospovirus/genética , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Mung bean yellow mosaic India virus (MYMIV) is a member of genus begomoviridae and its genome comprises of bipartite (two components, namely DNA-A and DNA-B), single-stranded, circular DNA of about 2.7 kb. During rolling circle replication (RCR) of the DNA, the stability of the genome and maintenance of the stem-loop structure of the replication origin is crucial. Hence the role of host single-stranded DNA-binding protein, Replication protein A (RPA), in the RCR of MYMIV was examined. Two RPA subunits, namely the RPA70 kDa and RPA32 kDa, were isolated from pea and their roles were validated in a yeast system in which MYMIV DNA replication has been modelled. Here, we present evidences that only the RPA32 kDa subunit directly interacted with the carboxy terminus of MYMIV-Rep both in vitro as well as in yeast two-hybrid system. RPA32 modulated the functions of Rep by enhancing its ATPase and down regulating its nicking and closing activities. The possible role of these modulations in the context of viral DNA replication has been discussed. Finally, we showed the positive involvement of RPA32 in transient replication of the plasmid DNA bearing MYMIV replication origin using an in planta based assay.
Assuntos
DNA Helicases/metabolismo , DNA Viral/biossíntese , Proteínas de Ligação a DNA/metabolismo , Geminiviridae/genética , Proteína de Replicação A/metabolismo , Transativadores/metabolismo , Proteínas Virais/metabolismo , Bioensaio , Replicação do DNA , Teste de Complementação Genética , Mutação , Pisum sativum/genética , Pisum sativum/crescimento & desenvolvimento , Pisum sativum/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteína de Replicação A/genética , Nicotiana/virologia , Técnicas do Sistema de Duplo-Híbrido , Leveduras/genéticaRESUMO
Geminiviruses replicate by rolling circle mode of replication (RCR) and the viral Rep protein initiates RCR by the site-specific nicking at a conserved nonamer (TAATATT downward arrow AC) sequence. The mechanism of subsequent steps of the replication process, e.g. helicase activity to drive fork-elongation, etc. has largely remained obscure. Here we show that Rep of a geminivirus, namely, Mungbean yellow mosaic India virus (MYMIV), acts as a replicative helicase. The Rep-helicase, requiring > or =6 nt space for its efficient activity, translocates in the 3'-->5' direction, and the presence of forked junction in the substrate does not influence the activity to any great extent. Rep forms a large oligomeric complex and the helicase activity is dependent on the oligomeric conformation ( approximately 24mer). The role of Rep as a replicative helicase has been demonstrated through ex vivo studies in Saccharomyces cerevisiae and in planta analyses in Nicotiana tabacum. We also establish that such helicase activity is not confined to the MYMIV system alone, but is also true with at least two other begomoviruses, viz., Mungbean yellow mosaic virus (MYMV) and Indian cassava mosaic virus (ICMV).
Assuntos
Begomovirus/enzimologia , Begomovirus/genética , DNA Helicases/metabolismo , Replicação do DNA , Proteínas Virais/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , DNA Helicases/genética , DNA Viral/biossíntese , Deleção de Sequência , Proteínas Virais/genéticaRESUMO
Helicobacter pylori causes gastric ulcer diseases and gastric adenocarcinoma in humans. Not much is known regarding DNA replication in H.pylori that is important for cell survival. Here we report the cloning, expression and characterization of H.pylori DnaB (HpDnaB) helicase both in vitro and in vivo. Among the DnaB homologs, only Escherichia coli DnaB has been studied extensively. HpDnaB showed strong 5' to 3' helicase and ATPase activity. Interestingly, H.pylori does not have an obvious DnaC homolog which is essential for DnaB loading on the E.coli chromosomal DNA replication origin (oriC). However, HpDnaB can functionally complement the E.coli DnaB temperature-sensitive mutant at the non-permissive temperature, confirming that HpDnaB is a true replicative helicase. Escherichia coli DnaC co-eluted in the same fraction with HpDnaB following gel filtration analysis suggesting that these proteins might physically interact with each other. It is possible that a functional DnaC homolog is present in H.pylori. The complete characterization of H.pylori DnaB helicase will also help the comparative analysis of DnaB helicases among bacteria.