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PsiI, a unique Restriction Endonuclease Recognizing the DNA Sequence 5'-TTA^TAA-3'

 

This email address is being protected from spambots. You need JavaScript enabled to view it. , O. A. Belichenko, N. A. Lebedeva, and S. Kh. Degtyarev

Translated from BIOCHEMISTRY (Russia) Vol. 64, No 4, pp. 481 - 482, 1999

 

PsiI, a new restriction endonuclease produced by the bacterial strain Pseudomonas sp. SE-G49, has been isolated and characterized. The enzyme cleaves DNA in the middle of its palindromic recognition sequence 5-TTA^TAA-3'. Thus, PsiI belongs to a rare group of type II restriction endonucleases whose recognition sites consist of AT base pairs only

 

Restriction endonucleases are widely used in molecular biology. Since the discovery of this group of enzymes, more than 200 prototypes of site-specific type II endonucleases have been characterized; 56 enzymes recognize palindromic sequences consisting of six nucleotides [1]. There are only few enzymes whose recognition sites consist of AT base pairs only. Earlier, only three such restriction endonuclease prototypes were described: DraI (TTT^AAA) [2], SspI (AAT^ATT) (Schildkraut I and Grandoni R., unpublished data), and VspI (AT^TAAT) [3].
We have isolated a new restriction endonuclease named PsiI. PsiI is produced by the bacterial strain Pseudomonas species SE-G49 and recognizes the nucleotide sequence 5’-TTATAA-3’.

 

MATERIALS AND METHODS

The producing strain P. species SE-G49 (SibEnzyme Ltd. collection of microorganisms) was grown in modified Luria medium containing 10 g/liter trypton, 5 g/liter yeast extract, and 1 g/liter NaCl, at pH 7.2 and 30°C with aeration. Cells were harvested by centrifugation when the culture was in the late logarithmic stage.
The resulting biomass was suspended in buffer containing 10 mM Tris-HCl, pH 7.6, 0.1 mM EDTA, 7 mM mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 0.01% Triton X-100. The cells were disrupted by ultrasonication (7 times for 45 sec) using a Soniprep 150 disintegrator (MSE, UK) at 1-min intervals to allow the suspension to be cooled in an ice bath.
The PsiI restriction endonuclease was isolated by consecutive chromatographies on phosphocellulose P11 (Whatman, UK), hydroxyapatite (BioRad, USA), and heparin-Sepharose (Sigma, USA).
DNAs was hydrolyzed by the PsiI endonuclease in 50 μl of reaction mixture containing 50 mM Tris-HCl (pH 7.6, 25°C), 10 mM MgCl2, 100 mM NaCl, and 20 μg/ml DNA in which 1 μl of the enzyme preparation was added. After a 1-h incubation at 37°C, the reaction products were separated by electrophoresis in 1% agarose in Tris-borate buffer containing 89 mM Tris, 89 mM H3BO4, and 2 mM Na-EDTA (pH 8.3).
32P-labeled oligonucleotide (Fig.1) with a corresponding complementary strand was used to determine the phosphodiester bond to be hydrolyzed. The oligonucleotide duplex was cleaved by restriction endonucleases under the conditions indicated by the producers. The products of hydrolysis were separated by electrophoresis in 20% polyacrylamide gel in the Tris-borate buffer in the presence of 7 M urea.
The DNA preparations, oligonucleotides, and restriction endonucleases used in this work were from SibEnzyme Ltd. (Novosibirsk, Russia).

Fig. 1. Autoradiogram of the electrophoresis of the oligonucleotide cleavage products separation by Psil and other restriction endonucleases. Restriction endonudeases: 1) AclWI; 2) AclWI + PsiI; 3) PsiI; 4) Sse9I + PsiI: 5) Sse9I; 6) Bme18I. 7) Uncleaved duplex. The sequence of the labeled chain of the duplex, the recognition sites of the restriction endonucleases, and the cleavage points are indicated on the left.

 

 

Fig. 1. Autoradiogram of the electrophoresis of the oligonucleotide cleavage products separation by Psil and other restriction endonucleases.

Restriction endonudeases: 1) AclWI; 2) AclWI + PsiI; 3) PsiI; 4) Sse9I + PsiI: 5) Sse9I; 6) Bme18I. 7) Uncleaved duplex. The sequence of the labeled chain of the duplex, the recognition sites of the restriction endonucleases, and the cleavage points are indicated on the left.



 

 

Fig. 2. Electrophoresis of the products of hydrolysis of various DNA preparations by PsiI restriction endonuclease. DNA: 7) lambda phage; 2) T7 phage; 3) type 2 adenovirus. M, length markers (fragments of lambda DNA/BmeI8I)

 

 

Fig. 2. Electrophoresis of the products of hydrolysis of various DNA preparations by PsiI restriction endonuclease.

DNA: 7) lambda phage; 2) T7 phage; 3) type 2 adenovirus. M, length markers (fragments of lambda DNA/BmeI8I)

 

 

 

RESULTS AND DISCUSSION

We have isolated PsiI, a novel site-specific endonuclease, from strain Pseudomonas. species SE-G49. The maximal enzymatic activity is found in the B buffer. PsiI is not thermostable; it is inactivated by incubation at 65°C for 20 min.
The numbers and the lengths of DNA fragments produced by PsiI in the course of substrate DNAs cleavage (Fig. 2) correspond to those theoretically predicted for a restriction endonuclease which recognizes nucleotide sequence 5'-TTATAA-3'. Thus, PsiI is a new restriction endonuclease prototype specific for AT nucleotide sequences.
The positions of DNA cleavage by PsiI endonuclease were determined by comparison of the lengths of the oligonucleotide duplex fragments produced by this enzyme and by some other restriction endonucleases (Fig. 1). The data indicate that PsiI cleaves the substrate in the middle of its recognition sequence, forming "blunt" ends of the DNA fragments.
The suggested method of enzyme purification yields PsiI restriction endonuclease preparation which is free of phosphatase and endo- and exonuclease activities. Thus, this new enzyme can be used in genetic engineering and molecular biological studies.

The authors are grateful to D. A. Gonchar and T. A. Madina for their kind assistance in experimental work.

 

REFERENCES

  1. Roberts, R. J., and Macelis, D. (1998) Nucleic Acids Res., 26, 338-350.
  2. Purvis, I. J., and Moseley, B. E. B. (1983) Nucleic Acids Res., 11, 5467-5474.
  3. Degtyarev, S. Kh., Repin, V. E., Rechkunova, N. I., Chizhikov, V. E., Malygin, E. G, Mikhailov, V. V., and Rasskazov, V. A. (1987) Bioorg. Khim., 13, 420-421.