RESUMO
Resolution of Holliday junctions into separate DNA duplexes requires enzymatic cleavage of an equivalent strand from each contributing duplex at or close to the point of strand exchange. Diverse Holliday junction-resolving enzymes have been identified in bacteria, bacteriophages, archaea and pox viruses, but the only eukaryotic examples identified so far are those from fungal mitochondria. We have now determined the crystal structure of Ydc2 (also known as SpCce1), a Holliday junction resolvase from the fission yeast Schizosaccharomyces pombe that is involved in the maintenance of mitochondrial DNA. This first structure of a eukaryotic Holliday junction resolvase confirms a distant evolutionary relationship to the bacterial RuvC family, but reveals structural features which are unique to the eukaryotic enzymes. Detailed analysis of the dimeric structure suggests mechanisms for junction isomerization and communication between the two active sites, and together with site-directed mutagenesis identifies residues involved in catalysis.
Assuntos
Cristalografia por Raios X , Endodesoxirribonucleases/química , Mitocôndrias/enzimologia , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimologia , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Domínio Catalítico , Clonagem Molecular , Dimerização , Evolução Molecular , Isomerismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fases de Leitura Aberta , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Secundária de Proteína , Homologia de Sequência de AminoácidosRESUMO
The thermodynamic parameters affecting protein-protein multimeric self-assembly equilibria of the histone-like protein H-NS were quantified by "large zone" gel-permeation chromatography. The abundance of the different association states (monomer, dimer, and tetramer) were found to be strictly dependent on the monomeric concentration and affected by physical (temperature) and chemical (cations) parameters. On the basis of the results obtained in this study and the available structural information concerning this protein, a mechanism is proposed to explain the association behavior also in relation to the functional properties of the protein.
Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Calorimetria , Cátions Monovalentes/farmacologia , Cromatografia em Gel , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/metabolismo , Cinética , Substâncias Macromoleculares , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , TermodinâmicaRESUMO
A multiposition microdialysis system suitable for simultaneous microsample applications (between 10 microL and 500 microL), has been developed. Each sample, contained in a specially designed microfuge dialysis tube (mDT), is dialysed independently from the other samples. Each mDT has its own membrane, and this feature allows the use of different membranes and dialysis times for different samples. The microdialysis apparatus is kept at constant temperature by an external thermostat, avoiding the use of a cold box. The dialysis release time for small ions, a parameter used for quantitation of microdialysis efficiency, decreases from 22.9 min (for a 200 microL sample) to 7 min (for a 50 microL sample). The sample is efficiently recovered by centrifugation. Quantitative recoveries (90%) of different proteins and DNA were achieved after microdialysis by mDT.
