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Crystals of site-speci®c 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 Ê 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, = 110.1. There is one molecule in the asymmetric unit and the solvent content is estimated to be 53% by volume.

Three site-specific endonucleases were found in thermophilic strain Bacillus species D6. One of them, BspD6II, is an isoschizomer of Eco57I. The second, BspD6III, is present in the strain in very small amount; therefore, it has not been characterized. This paper is devoted to the third, BspD6I, which recognizes pentanucleotide site 5'-GAGTC-3' and cleaves only one DNA strand at a distance of 4 nucleotides from the site in the 3'-direction in the chain with the GAGTC sequence, i.e., it behaves as a site-specific nickase. Nickase N.BspD6I cleaves one DNA strand only in double-stranded DNA and does not cleave single-stranded DNA. Site-specific methylase SscL1I (an isohypectomer of M*HinfI) that methylates adenine in the sequence 5'-GAGTC-3' prevents DNA hydrolysis by nickase BspD6I.

A new method for hybridization analysis of nucleic acids is proposed on the basis of the ability of site-specific nickases to cleave only one DNA strand. The method is based on the use of a labeled oligonucleotide with the recognition site of the nickase hybridized with the target (DNA or RNA) at an optimal temperature of the enzyme (55 degrees C). The two shorter oligonucleotides formed after the cleavage with the nickase do not complex with the target. Thus, a multiple cleavage of the labeled oligonucleotide takes place on one target molecule. The cleavage of the nucleotide is recorded either by polyacrylamide gel electrophoresis (when a radioactive labeled oligonucleotide is used) or by fluorescence measurements (if the oligonucleotide has the structure of a molecular beacon). The new method was tested on nickase BspD6I and a radioactive oligonucleotide complementary to the polylinker region of the viral DNA strand in bacteriophage M13mp19. Unfortunately, nickase BspD6I does not cleave DNA in the RNA-DNA duplexes and therefore cannot be used for detection of RNA targets.

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.

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.