Site-specific endonuclease NspLKI is an isoschizomer of endonuclease HaeIII

Site-specific endonuclease NspLKI has been isolated and purified to functionally pure state from soil bacterium Nocardia species LK by successive chromatography on columns with phosphocellulose, HTP hydroxyapatite, and heparin-Sepharose. The isolated enzyme recognizes the 5'-GG downward arrowCC-3' sequence on DNA and cleaves it as indicated by the arrow, i.e., it is an isoschizomer of HaeIII. The final enzyme yield is 1.105 units per gram of wet biomass. The enzyme is active in the temperature range of 25-60 degrees C with an optimum at 48-55 degrees C; it does not lose activity on storage for three days at room temperature. An optimal buffer is HRB containing 10 mM Tris-HCl, pH 7.4, 200 microgram/ml albumin, 10 mM MgCl2, and 100 mM NaCl.     

PCR fragmentation of DNA

A method has been developed to prepare random DNA fragments using PCR. First, two cycles are carried out at 16 degrees C with the Klenow's fragment and oligonucleotides (random primers) with random 3'-sequences and the 5'-constant part containing the site for cloning with the site-specific endonuclease. The random primers can link to any DNA site, and random DNA fragments are formed during DNA synthesis. During the second cycle, after denaturation of the DNA and addition of the Klenow's fragment, the random primers can link to newly synthesized DNA strands, and after DNA synthesis single-stranded DNA fragments are produced which have a constant primer sequence at the 5'-end and a complementary to it sequence at the 3'-end. During the third cycle, the constant primer is added and double-stranded fragments with the constant primer sequences at both ends are formed during DNA synthesis. Incubation for 1 h at 37 degrees C degrades the oligonucleotides used at the first stage due to endonuclease activity of the Klenow's fragment. Then routine PCR amplification is carried out using the constant primer. This method is more advantageous than hydrodynamic methods of DNA fragmentation widely used for "shotgun" cloning.     

Bacillus species LU4 is an effective producer of thermostable site-specific endonuclease BspLU4I,an isoschizomer of AvaI

The site-specific endonuclease BspLU4I was discovered in the thermophilic Bacillus species LU4 strain and purified to functionally pure state by chromatography on blue agarose, hydroxyapatite HTP, and heparin-Sepharose columns. Analysis of cleavage patterns of different DNAs with known nucleotide sequences demonstrated that the enzyme recognizes the CPyCGPuG site on the DNA. Cleavage points in the sequence were determined with the elongated primer method. It was shown that the endonuclease is an isoschizomer of AvaI. The final yield of the enzyme is 2.25·10⁶ units per g wet biomass.     

N.BspD6I DNA nickase strongly stimulates template-independent synthesis of non-palindromic repetitive DNA by Bst DNA polymerase

Highly efficient DNA synthesis without template and primer DNAs occurs when N.BspD6I DNA nickase is added to a reaction mixture containing deoxynucleoside triphosphates and the large fragment of Bst DNA polymerase. Over a period of 2 h, virtually all the deoxynucleoside triphosphates (dNTPs) become incorporated into DNA. Inactivation of N.BspD6I nickase by heating inhibits DNA synthesis. Optimal N.BspD6I activity is required to achieve high yields of synthesized DNA. Electron microscopy data revealed that the majority of DNA molecules have a branched structure. Cloning and sequencing of the fragments synthesized demonstrated that the DNA product mainly consists of multiple hexanucleotide non-palindromic tandem repeats containing nickase recognition sites. A possible mechanism is discussed that addresses template-independent DNA synthesis stimulated by N.BspD6I nickase.     

Location of the Bases Modified by M.<Emphasis Type="Italic">Bco</Emphasis>KIA and M.<Emphasis Type="Italic">Bco</Emphasis>KIB Methylases in the Sequence 5′-CTCTTC-3′/5′-GAAGAG-3′

The strain Bacillus coagulans K contains two DNA-methyltransferases, M.BcoKIA and M.BcoKIB, which recognize the sequence 5 -CTCTTC-3 /5 -GAAGAG-3 and possess N4-methylcytosine and N6-methyladenine specificities, respectively. A special construct containing the recognition site of BcoKI and sites of four IIS restriction endonucleases (IIS restriction endonuclease cassette) was designed to locate the nucleotides modified by the methylases. The modified bases were determined as: 5 -m(4)CTCTTC-3 /5 -GAAGAm(6)G-3 .     

Restriction Endonuclease BspD6II,a New Thermophilic Isoschizomer of Eco57I

Russian Journal of Bioorganic Chemistry - From the Bacillus species D6 strain, the restriction endonuclease BspD6II has been isolated and characterized. It recognizes the asymmetric region...     

Nicking endonucleases

Nicking endonucleases are a new type of enzymes. Like restriction endonucleases, they recognize short specific DNA sequence and cleave DNA at a fixed position relatively to the recognition sequence. However, unlike restriction endonucleases, nicking endonucleases cleave only one predetermined DNA strand. Until recently, nicking endonucleases were suggested to be naturally mutated restriction endonucleases which had lost their ability to dimerize and as a result the ability to cleave the second strand. We have shown that nicking endonucleases are one of the subunits of heterodimeric restriction endonucleases. Mechanisms used by various restriction endonucleases for double-stranded cleavage, designing of artificial nicking endonucleases on the basis of restriction endonucleases, and application of nicking endonucleases in molecular biology are reviewed.     

A site-specific endonuclease from the thermophilic strain Bacillus species F4.

A strain producing the site-specific endonuclease BspF4I was found during screening of thermophilic bacteria isolated from soil. The restriction endonuclease, free from contaminant nonspecific nucleases, was purified using three steps of column chromatography--on hydroxyapatite, blue agarose, and DEAE-Trisacryl. The enzyme is stable on storage and exhibits maximal activity at 48-56 degrees C in the presence of albumin in buffer containing 10 mM Tris-HCl (pH 7.5) and 10 mM MgCl2. BspF4I recognizes the sequence 5;-GGNCC-3; on DNA and is an isomer and not an isoschizomer of the endonuclease Sau96I. Unlike the prototype, BspF4I does not cleave the site in a defined way. A strand with purine in the center of the sequence is cleaved after the first G, as in the case of the prototype, while the strand with pyrimidine is cleaved either before or after the first G.