RESUMO
Before cleaving DNA substrates with two recognition sites, the Cfr10I, NgoMIV, NaeI and SfiI restriction endonucleases bridge the two sites through 3D space, looping out the intervening DNA. To characterise their looping interactions, the enzymes were added to plasmids with two recognition sites interspersed with two res sites for site-specific recombination by Tn21 resolvase, in buffers that contained either EDTA or CaCl2 so as to preclude DNA cleavage by the endonuclease; the extent to which the res sites were sequestered into separate loops was evaluated from the degree of inhibition of resolvase. With Cfr10I, a looped complex was detected in the presence but not in the absence of Ca(2+); it had a lifetime of about 90 seconds. Neither NgoMIV nor NaeI gave looped complexes of sufficient stability to be detected by this method. In contrast, SfiI with Ca(2+) produced a looped complex that survived for more than seven hours, whereas its looping interaction in EDTA lasts for about four minutes. When resolvase was added to a SfiI binding reaction in EDTA followed immediately by CaCl2, the looped DNA was blocked from recombination while the unlooped DNA underwent recombination. By measuring the distribution between looped and unlooped DNA at various SfiI concentrations, and by fitting the data to a model for DNA binding by a tetrameric protein to two sites in cis, an equilibrium constant for the looping interaction was determined. The equilibrium constant was essentially independent of the length of DNA between the SfiI sites.
Assuntos
DNA/química , DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Conformação de Ácido Nucleico , Transposon Resolvases , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cálcio/metabolismo , DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Dosagem de Genes , Cinética , Plasmídeos/química , Plasmídeos/genética , Plasmídeos/metabolismo , Recombinação Genética/genética , Sequências Repetitivas de Ácido Nucleico/genética , Especificidade por Substrato , Temperatura , TermodinâmicaAssuntos
Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Proteínas de Bactérias/química , Sítios de Ligação , DNA/metabolismo , Metilação de DNA , Desoxirribonucleases de Sítio Específico do Tipo II/classificação , Especificidade por SubstratoRESUMO
Prevotella intermedia and the newly described P. nigrescens cannot be reliably distinguished by phenotypic tests. In this study, restriction endonuclease digestion of amplified 16S rDNA (16S rDNA PCR-RFLP) was used to generate restriction profiles of the type strains of P. intermedia and P. nigrescens and 43 fresh isolates identified as belonging to one of the two species. Whole-cell protein profiles were obtained by SDS-PAGE for comparative purposes. The type strains of P. intermedia and P. nigrescens were easily distinguished by 16S rDNA PCR-RFLP and the fresh isolates were assigned to either species on the basis of their restriction profiles. The identifications obtained were identical to those obtained by protein profiles. 16S rDNA PCR-RFLP is a rapid and reliable method for the differentiation of P. intermedia and P. nigrescens.
