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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.