RESUMEN
The UmuC/DinB family of bypass polymerases is responsible for translesion DNA synthesis and includes the human polymerases eta, iota, and kappa. We determined the 2.3 A resolution crystal structure of a catalytic fragment of the DinB homolog (Dbh) polymerase from Sulfolobus solfataricus and show that it is nonprocessive and can bypass an abasic site. The structure of the catalytic domain is nearly identical to those of most other polymerase families. Homology modeling suggests that there is minimal contact between protein and DNA, that the nascent base pair binding pocket is quite accessible, and that the enzyme is already in a closed conformation characteristic of ternary polymerase complexes. These observations afford insights into the sources of low fidelity and low processivity of the UmuC/DinB polymerases.
Asunto(s)
Proteínas Arqueales/química , Proteínas Bacterianas/química , Dominio Catalítico , ADN Polimerasa Dirigida por ADN/química , Proteínas de Escherichia coli , Sulfolobus/enzimología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cristalografía por Rayos X , ADN/metabolismo , ADN Polimerasa beta/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Genes Reporteros/genética , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Sulfolobus/química , Moldes GenéticosRESUMEN
Underpinned by a database of more than a dozen different crystal structures, an increasingly complete and coherent picture of polymerase structure and function is emerging. Recently determined structures of DNA and RNA polymerases have revealed some of the molecular features and structural changes governing catalysis, oligomerization, processivity and fidelity. Despite having minimal similarities in sequence and protein topology, the polymerases all display a functionally analogous set of subdomains that bind the primer, template and nucleotide substrates in similar though not identical fashions. The two-metal-ion mechanism for nucleotide incorporation, however, is shared even by nonhomologous polymerases.
Asunto(s)
ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/metabolismo , Secuencia de Bases , Dominio Catalítico , ADN/química , ADN/metabolismo , Humanos , Modelos Moleculares , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Conformación de Ácido Nucleico , Conformación Proteica , ARN/química , ARN/metabolismo , Especificidad por SustratoRESUMEN
Using a hairpin primer/template RNA derived from sequences present at the 3' end of the poliovirus genome, we investigated the RNA-binding and elongation activities of highly purified poliovirus 3D polymerase. We found that surprisingly high polymerase concentrations were required for efficient template utilization. Binding of template RNAs appeared to be the primary determinant of efficient utilization because binding and elongation activities correlated closely. Using a three-filter binding assay, polymerase binding to RNA was found to be highly cooperative with respect to polymerase concentration. At pH 5.5, where binding was most cooperative, a Hill coefficient of 5 was obtained, indicating that several polymerase molecules interact to retain the 110-nt RNA in a filter-bound complex. Chemical crosslinking with glutaraldehyde demonstrated physical polymerase-polymerase interactions, supporting the cooperative binding data. We propose a model in which poliovirus 3D polymerase functions both as a catalytic polymerase and as a cooperative single-stranded RNA-binding protein during RNA-dependent RNA synthesis.
Asunto(s)
Poliovirus/enzimología , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , ARN Polimerasa Dependiente del ARN/metabolismo , Secuencia de Bases , Reactivos de Enlaces Cruzados , Expresión Génica , Modelos Genéticos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Poliovirus/genética , Unión Proteica , Conformación Proteica , ARN Polimerasa Dependiente del ARN/genética , ARN Polimerasa Dependiente del ARN/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
We are developing an in vitro system for poliovirus RNA recombination. In this system, two mutant RNAs are replicated with poliovirus RNA-dependent RNA polymerase. Recombination will produce RNAs containing neither mutation and will be the only progeny RNAs that are infectious. We will use this system to determine what proteins and reaction conditions are required for recombination and to study the details of the mechanism of recombination.
Asunto(s)
Poliovirus/genética , ARN Viral/genética , Recombinación Genética , Células HeLa , Mutagénesis , ARN Polimerasa Dependiente del ARN/metabolismo , Moldes Genéticos , TransfecciónRESUMEN
We have isolated several Tetrahymena thermophila chromosomal DNA fragments which function as autonomously replicating sequences (ARS) in the heterologous Saccharomyces cerevisiae and Schizosaccharomyces pombe selection systems. The Tetrahymena ARS sequences were first isolated in S. cerevisiae and were derived from non-ribosomal micro- and macronuclear DNA. Sequence analysis of the ARS elements identified either perfect or close matches with the 11 bp S. cerevisiae ARS core consensus sequence. Subcloning studies of two Tetrahymena ARS elements defined functional regions ranging in size from 50 to 300 bp. Testing of the ARS elements in S. pombe revealed that most of the T. thermophila inserts confer ARS function in both yeasts, at least in the sense of promoting a high transformation frequency to plasmids which contain them. However, the actual sequences responsible for ARS activity were not always identical in the two yeasts.
Asunto(s)
Replicación del ADN , ADN/fisiología , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Schizosaccharomyces/genética , Tetrahymena/genética , Transformación Genética , Animales , Secuencia de Bases , Clonación Molecular , ADN/aislamiento & purificación , Datos de Secuencia MolecularRESUMEN
Partial N-terminal amino acid sequences for the three largest nonstructural proteins of two flaviviruses, yellow fever virus and St. Louis encephalitis virus, have been obtained. The determined sequences of these proteins exhibit significant amino acid sequence homology, and allow the positioning of these three nonstructural proteins in the polyprotein sequence deduced from the nucleotide sequence of yellow fever virus (C. M. Rice, E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, and J. H. Strauss, 1985, Science 229, 726-733.) The deduced start points support the hypothesis that the N terminus of nonstructural glycoprotein NS1 results from cleavage by signalase, whereas the N termini of NS3 and NS5 result from cleavages following double basic residues that are flanked by amino acids with short side chains.