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Abstract

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.

Nicking endonucleases

“…However, they are lost during subsequent purification. Owing to this, researchers stopped further work with such endonucleases [4,18,20]. Thus, at initial stages of nickase isolation directly from the strain Bacillus sp.…”

“…Thus, at initial stages of nickase isolation directly from the strain Bacillus sp. D6, we came into collision with the fact that restriction endonuclease called by us BspD6III was eluted from the first column in addition to nickase [4]. However, during further purification of this endonu clease only nickase was eluted from the column instead of endonuclease.…”

“…In 2001 nick ase N.BspD6I was isolated in Laboratory of N. I. Matvienko from strain D6 of Bacillus sp. [4]. In the same year, Dedkov et al [5] described a nicking endonuclease N.Bst9I isolated from B. stearothermophilus strain 9.…”

Crystallization and preliminary crystallographic analysis of the site-specific DNA nickase Nb.BspD6I

“…The Nb.BspD6I nickase (Zheleznaya et al, 2001) has been identi®ed in Bacillus sp. strain D6 isolated from soil.…”

Oligomerization of site-specific nicking endonuclease BspD6I at high protein concentrations

“…In this study we investigated NE BspD6I (Nt.BspD6I) found in original thermophilic strain of Bacillus species D6 [8]. The protein Nt.BspD6I is one of two subunits of a heterodimeric restriction endonu clease R.BspD6I, which includes also the second small subunit [9].…”