ABSTRACT
Ability of site-specific nickase BspD6I (Nt.BspD6I) to oligomerize at concentrations > or = 0.5 microM (> or = 0.035 mg/mL) is studied. Three states of Nt.BspD6I are registered via electrophoretic studies both in the presence and in the absence of DNA. Estimation of their molecular mass allows assigning them as a monomer, a dimer and a trimer. Both dimeric and monomeric Nt.BspD6I are shown to hydrolyze its DNA substrate with the identical specificity. Calculation of the electrostatic potential distribution on the Nt.BspD6I globule surface shows that the protein molecule is a dipole. The Nt. BspD6I oligomeric forms are likely to be the result of ionic protein interactions.
Subject(s)
DNA-Binding Proteins/chemistry , Deoxyribonuclease I/chemistry , Protein Structure, Tertiary , Bacillus/enzymology , DNA/chemistry , Protein MultimerizationABSTRACT
We are the first to have isolated a protein (186 amino acid residues) encoded by the open reading frame adjacent to the end of the BspD6I nickase (N.BspD6I) gene. Cleavage of both DNA strands near the sequence recognized by nickase (5 -GAGTC/5 -GACTC) occurs when this protein is added to the reaction mixture containing N.BspD6I. The protein encoded by the open reading frame and the nickase are suggested to be subunits of heterodimeric restriction endonuclease R.BspD6I.
Subject(s)
DNA Restriction Enzymes/genetics , Deoxyribonuclease I/genetics , Escherichia coli Proteins/genetics , Amino Acid Sequence , DNA Restriction Enzymes/isolation & purification , DNA Restriction Enzymes/metabolism , Deoxyribonuclease I/metabolism , Dimerization , Escherichia coli Proteins/metabolism , Models, Genetic , Molecular Sequence Data , Open Reading Frames , Protein Subunits/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolismABSTRACT
The gene of methylase M.SccL1I that protects DNA against hydrolysis with the nickase N.BspD6I was inserted into plasmid pRARE carrying genes of tRNA, which are rare in E. coli. The insertion of the gene sscML1I into pRARE was reasoned by incompatibility of pRARE and the plasmid carrying the gene sscML1I, because both plasmids contained the same ori-site. Upon transformation of E. coli TOP10F cells with both the recombinant plasmid pRARE/MSsc and the expression vector pET28b containing the nickase gene bspD6IN under the phage T7 promoter, a strain of E. coli was obtained which produced 7 x 10(5) units of the nickase N.BspD6I per 1 g wet biomass, and this yield was two orders of magnitude higher than the yield of the enzyme from the strain free of pRARE/MSsc.
Subject(s)
Bacillus/enzymology , Endodeoxyribonucleases/genetics , Genetic Vectors , Amino Acid Sequence , Cloning, Molecular , Endodeoxyribonucleases/biosynthesis , Molecular Sequence Data , Plasmids , RNA, Transfer/geneticsABSTRACT
A fragment of chromosomal DNA from Bacillus species D6 containing the gene of nickase N.BspD6I and the regions adjacent to its 5;- and 3;-ends was cloned and sequenced. The nucleotide sequence of the nickase gene, except of one neutral change, is homologous to the nicking endonuclease N.BstNBI gene sequenced by Higgens et al. (2001). After integration of a PCR-copy of the nickase gene into an expression vector pET28b under the control of the phage T7 promoter, specific nicking activity was detected in the lysates of transformed E. coli cells.
