RESUMEN
We have cloned the genes encoding the chaperones of Meiothermus ruber, Hsp70 (Mru.Hsp70), Hsp40 (Mru.Hsp40) and Hsp22 (Mru.Hsp22). The genes hsp70, hsp22 and hsp40 of M. ruber are organized into an operon. The amino acid sequences of the three M. ruber chaperones show strong similarity with the heat shock proteins of Thermus thermophilus. Both Mru.Hsp40 and its homolog from T. thermophilus lack a cysteine-rich region. However, recombinant Mru.Hsp70 and Mru.Hsp40 associate in an ATP-dependent manner, and assemble into a complex in the absence of other proteins, unlike their counterparts from T. thermophilus, which require DafA for assembly. The analysis revealed that Mru.Hsp70 and Mru.Hsp40 assemble as monomers into the complex, although their homologs from T. thermophilus enter the complex as trimers. The Mru.Hsp70 and Mru.Hsp40 complex increases the spontaneous rate of refolding of denatured mitochondrial malate dehydrogenase by tenfold.
Asunto(s)
Genes Bacterianos/genética , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/genética , Thermus/química , Thermus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , ADN Bacteriano/química , ADN Bacteriano/genética , Expresión Génica , Proteínas HSP70 de Choque Térmico/metabolismo , Datos de Secuencia Molecular , Operón , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Thermus/clasificación , Thermus thermophilus/química , Thermus thermophilus/genéticaRESUMEN
Substrate-assisted catalysis was suggested to be involved in the DNA cleavage reaction of the restriction endonucleases (ENases) EcoRI and EcoRV, because experimental evidence exists that the phosphate group 3' to the scissile bond serves to deprotonate the attacking water. Here, we have addressed the question whether this is a general mechanistic feature of the reactions catalyzed by ENases. For this purpose, the cleavage rates of modified and unmodified oligodeoxyribonucleotides (oligos), in which the phosphate group 3' to the scissile bond is substituted by a methyl phosphonate, were measured for 17 enzymes. Only five turned out not to be inhibited by this modification (BglII, BstI, BstYI, Cfr10I and MunI); all others cleave the modified substrate at a strongly reduced rate or not at all. By employing a hemisubstituted oligo substrate we were able to further investigate the mechanism of inhibition of the latter group of ENases. Some of them cleave the unmodified strand of the modified substrate with a nearly unaltered rate, whereas the modified strand is cleaved very slowly or not at all (BamHI, Bsp143I, Eco72I, MflI, NdeII, Sau3AI, XhoII). The others (AluI, Cfr9I, DpnII, MboI, PvuII) cleave the modified strand of the modified substrate with a largely reduced rate or not at all. These ENases, however, cleave the unmodified strand with a reduced rate, too. Based on these results we conclude that BamHI, Bsp143I, Cfr9I, DpnII, Eco72I, MboI, MflI, NdeII, PvuII, Sau3AI and XhoII may possibly employ substrate assistance in catalysis.
Asunto(s)
Enzimas de Restricción del ADN/metabolismo , ADN/metabolismo , Oligodesoxirribonucleótidos/química , Oligodesoxirribonucleótidos/metabolismo , Secuencia de Bases , Sitios de Unión , Catálisis , Cinética , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
The Cfr9I restriction endonuclease recognizes and cleaves duplex DNA sequence C decreases CCGGG. The binding of restriction endonuclease Cfr9I to DNA was examined in the absence of Mg2+ using gel-mobility-shift and nitrocellulose-filter-binding assays. It was shown that restriction endonuclease Cfr9I bound DNA fragments either containing or lacking the canonical recognition sequence with equal affinity. These results suggest that the specificity of restriction endonuclease Cfr9I is expressed during the catalytic step. The cleavage of supercoiled pUC18 DNA by restriction endonuclease Cfr9I showed that at low concentrations of MgCl2, only with open-circular DNA, nicks appeared in one strand at the recognition sequence, while the cleavage of the second strand was very slow. At higher concentrations of MgCl2 the enzyme cleaves either one or both strands of the DNA. Under these conditions the supercoiled DNA was converted to open-circular and linear forms simultaneously rather than consecutively. It was shown that open-circular DNA was a poor substrate for restriction endonuclease Cfr9I. These results suggested that both Mg2+ and intact recognition sequence are required to drive the enzyme into correct conformation to ensure DNA cleavage.
Asunto(s)
ADN/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Secuencia de Bases , Sitios de Unión , Catálisis , ADN Circular/metabolismo , ADN Superhelicoidal/metabolismo , Hidrólisis , Cinética , Cloruro de Magnesio/farmacología , Datos de Secuencia Molecular , Plásmidos , Especificidad por SustratoRESUMEN
Chemical modification studies were performed to elucidate the role of Cys-residues in the catalysis/binding of restriction endonuclease Cfr9I. Incubation of restriction endonuclease Cfr9I with N-ethylmaleimide (NEM), iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) at pH 7.5 led to a complete loss of the catalytic activity. However, no enzyme inactivation was detectable after modification of the enzyme with iodoacetamide and methyl methanethiosulfonate. Complete protection of the enzyme against inactivation by NEM was observed in the presence of substrate implying that Cys-residues may be located at or in the vicinity of the active site of enzyme. Direct substrate-binding studies of native and modified restriction endonuclease Cfr9I using a gel-mobility shift assay indicated that the modification of the enzyme by NEM was hindered by substrate binding. A single Cys-residue was modified during the titration of the enzyme with DTNB with concomitant loss of the catalytic activity. The pH-dependence of inactivation of Cfr9I by NEM revealed the modification of the residue with the pKa value of 8.9 +/- 0.2. The dependence of the reaction rate of substrate hydrolysis by Cfr9I versus pH revealed two essential residues with pKa values of 6.3 +/- 0.15 and 8.7 +/- 0.15, respectively. The evidence presented suggests that the restriction endonuclease Cfr9I contains a reactive sulfhydryl residue which is non-essential for catalysis, but is located at or near the substrate binding site.