RESUMO
The mutated nickase Nt.BspD6I E418A has been obtained by site-directed mutagenesis. The purified protein has been crystallized, and its spatial structure has been determined at 2.45 Å resolution. An analysis of the crystal structures of the wild-type and mutated nickase have shown that the elimination of a carboxyl group due to the E418A mutation initiates marked conformational changes in both the N-terminal recognition domain and the C-terminal catalytic domain of nickase and insignificantly affects its linker domain. This is supported by changes in the functional properties of mutated nickase: an increase in the oligomerization capacity in the presence of a substrate, a reduction in the capacity to bind a substrate, and complete loss of catalytic activity.
Assuntos
Desoxirribonuclease I/química , Desoxirribonuclease I/metabolismo , Domínio Catalítico/genética , Desoxirribonuclease I/genética , Mutagênese Sítio-Dirigida , Mutação/genéticaRESUMO
The heterodimeric restriction endonuclease R.BspD6I is composed of a small subunit with a cleavage site and a large subunit, containing a recognition domain and a cleavage domain, that may function separately as a monomeric nicking endonuclease. Here, the crystallization of the small subunit and diffraction data collection to 1.5 A resolution are reported.
Assuntos
Desoxirribonuclease I/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Cristalização , Enzimas de Restrição do DNA/química , Enzimas de Restrição do DNA/isolamento & purificação , Desoxirribonuclease I/isolamento & purificação , Dimerização , Proteínas de Escherichia coli/isolamento & purificação , Subunidades Proteicas/química , Difração de Raios XRESUMO
Crystals of site-specific DNA nickase Nb.BspD6I (of molecular weight 70.8 kDa) have been grown at 291 K using PEG 8000 as precipitant. The diffraction pattern of the crystal extends to 3.3 A resolution at 100 K. The crystal belongs to space group P2(1), with unit-cell parameters a = 57.76, b = 90.67, c = 71.71, beta = 110.1 degrees. There is one molecule in the asymmetric unit and the solvent content is estimated to be 53% by volume.
Assuntos
Desoxirribonuclease I/metabolismo , Bacillus/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Cristalização , Cristalografia por Raios X , Desoxirribonuclease I/química , Desoxirribonuclease I/isolamento & purificação , Cinética , Especificidade por Substrato , Difração de Raios XRESUMO
The heterodimeric restriction endonuclease R.BspD6I from Bacillus species D6 recognizes a pseudosymmetric sequence and cuts both DNA strands outside the recognition sequence. The large subunit, Nt.BspD6I, acts as a type IIS site-specific monomeric nicking endonuclease. The isolated small subunit, ss.BspD6I, does not bind DNA and is not catalytically active. We solved the crystal structures of Nt.BspD6I and ss.BspD6I at high resolution. Nt.BspD6I consists of three domains, two of which exhibit structural similarity to the recognition and cleavage domains of FokI. ss.BspD6I has a fold similar to that of the cleavage domain of Nt.BspD6I, each containing a PD-(D/E)XK motif and a histidine as an additional putative catalytic residue. In contrast to the DNA-bound FokI structure, in which the cleavage domain is rotated away from the DNA, the crystal structure of Nt.BspD6I shows the recognition and cleavage domains in favorable orientations for interactions with DNA. Docking models of complexes of Nt.BspD6I and R.BspD6I with cognate DNA were constructed on the basis of structural similarity to individual domains of FokI, R.BpuJI and HindIII. A three-helix bundle forming an interdomain linker in Nt.BspD6I acts as a rigid spacer adjusting the orientations of the spatially separated domains to match the distance between the recognition and cleavage sites accurately.
Assuntos
Domínio Catalítico , Desoxirribonuclease I/química , Desoxirribonuclease I/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/química , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Sequência de Aminoácidos , Catálise , DNA Bacteriano/metabolismo , Dimerização , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
We have developed a simple isothermal (55 degrees C) reaction that permits detection of DNA targets using only two components: a molecular beacon and a site-specific DNA nickase without deoxyribonucleotide triphosphates and primers. The loop sequence of the molecular beacon should contain a DNA nickase recognition site. The nickase-molecular beacon (NMB) combination permits a 100-fold increase in fluorescent signal. The applications of the NMB assay for enhancement of fluorescent signal in some isothermal methods are discussed.