RESUMO
Interest is growing for 3D models of the biological mesoscale, the intermediate scale between the nanometer scale of molecular structure and micrometer scale of cellular biology. However, it is currently difficult to gather, curate and integrate all the data required to define such models. To address this challenge we developed Mesoscope (mesoscope.scripps.edu/beta), a web-based data integration and curation tool. Mesoscope allows users to begin with a listing of molecules (such as data from proteomics), and to use resources at UniProt and the PDB to identify, prepare and validate appropriate structures and representations for each molecule, ultimately producing a portable output file used by CellPACK and other modeling tools for generation of 3D models of the biological mesoscale. The availability of this tool has proven essential in several exploratory applications, given the high complexity of mesoscale models and the heterogeneity of the available data sources.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Origem de Replicação , Vírus 40 dos Símios/genética , Vírus 40 dos Símios/imunologia , Virologia/métodos , Animais , Pegada de DNA/métodos , Replicação do DNA , DNA Viral/genética , DNA Viral/isolamento & purificação , DNA Viral/metabolismo , Eletroforese em Gel de Poliacrilamida , Hibridização de Ácido Nucleico , Oligonucleotídeos/isolamento & purificação , Análise de Sequência de DNA/métodos , Vírus 40 dos Símios/fisiologia , Replicação ViralRESUMO
Using subfragments of the simian virus 40 (SV40) core origin, we demonstrate that two alternative modules exist for the assembly of T-antigen (T-ag) double hexamers. Pentanucleotides 1 and 3 and the early palindrome (EP) constitute one assembly unit, while pentanucleotides 2 and 4 and the AT-rich region constitute a second, relatively weak, assembly unit. Related studies indicate that on the unit made up of pentanucleotide 1 and 3 and the EP assembly unit, the first hexamer forms on pentanucleotide 1 and that owing to additional protein-DNA and protein-protein interactions, the second hexamer is able to form on pentanucleotide 3. Oligomerization on the unit made up of pentanucleotide 2 and 4 and the AT-rich region is initiated by assembly of a hexamer on pentanucleotide 4; subsequent formation of the second hexamer takes place on pentanucleotide 2. Given that oligomerization on the SV40 origin is limited to double-hexamer formation, it is likely that only a single module is used for the initial assembly of T-ag double hexamers. Finally, we discuss the evidence that nucleotide hydrolysis is required for the remodeling events that result in the utilization of the second assembly unit.
Assuntos
Antígenos Transformantes de Poliomavirus/fisiologia , Origem de Replicação , Vírus 40 dos Símios/fisiologia , Proteínas do Core Viral/fisiologia , Montagem de Vírus , Trifosfato de Adenosina/química , Adenilil Imidodifosfato/química , Animais , Antígenos Transformantes de Poliomavirus/química , Sequência de Bases , DNA Viral/química , Modelos Biológicos , Dados de Sequência Molecular , Oligonucleotídeos/química , Ligação Proteica , Vírus 40 dos Símios/genética , Proteínas do Core Viral/químicaRESUMO
Cell cycle-dependent phosphorylation of simian virus 40 (SV40) large tumor antigen (T-ag) on threonine 124 is essential for the initiation of viral DNA replication. A T-ag molecule containing a Thr-->Ala substitution at this position (T124A) was previously shown to bind to the SV40 core origin but to be defective in DNA unwinding and initiation of DNA replication. However, exactly what step in the initiation process is defective as a result of the T124A mutation has not been established. Therefore, to better understand the control of SV40 replication, we have reinvestigated the assembly of T124A molecules on the SV40 origin. Herein it is demonstrated that hexamer formation is unaffected by the phosphorylation state of Thr 124. In contrast, T124A molecules are defective in double-hexamer assembly on subfragments of the core origin containing single assembly units. We also report that T124A molecules are inhibitors of T-ag double hexamer formation. These and related studies indicate that phosphorylation of T-ag on Thr 124 is a necessary step for completing the assembly of functional double hexamers on the SV40 origin. The implications of these studies for the cell cycle control of SV40 DNA replication are discussed.
Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Origem de Replicação , Vírus 40 dos Símios/fisiologia , Proteínas do Core Viral/metabolismo , Replicação Viral , Substituição de Aminoácidos , Animais , Antígenos Transformantes de Poliomavirus/química , Sequência de Bases , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Oligonucleotídeos , Fragmentos de Peptídeos , Fosforilação , Treonina/metabolismo , Proteínas do Core Viral/químicaRESUMO
The regions of the simian virus 40 (SV40) core origin that are required for stable assembly of virally encoded T antigen (T-ag) and the T-ag origin binding domain (T-ag-obd(131-260)) have been determined. Binding of the purified T-ag-obd(131-260) is mediated by interactions with the central region of the core origin, site II. In contrast, T-ag binding and hexamer assembly requires a larger region of the core origin that includes both site II and an additional fragment of DNA that may be positioned on either side of site II. These studies indicate that in the context of T-ag, the origin binding domain can engage the pentanucleotides in site II only if a second region of T-ag interacts with one of the flanking sequences. The requirements for T-ag double-hexamer assembly are complex; the nucleotide cofactor present in the reaction modulates the sequence requirements for oligomerization. Nevertheless, these experiments provide additional evidence that only a subset of the SV40 core origin is required for assembly of T-ag double hexamers.