RU2475534C1 - BACTERIA STRAIN Planomicrobium koreense 78K-PRODUCENT OF SITE-SPECIFIC ENDONUCLEASE PkrI - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: bacteria strain Planomicrobium koreense 78k, is extracted from soils and provides for production of site-specific endonuclease PkrI, which recognises and splits both chains of nucleotide sequence of DNA, which contains three or four C5-methylcytosine bases in recognition site 5'-GCNAGC-3' with formation of a single-nucleotide 3'-protruding end. The strain is deposited in the Russian National Collection of Industrial Microorganisms FGUP GosNIIgenetika under the number B-10627. The provided strain may be used for extraction of the new site-specific endonuclease PkrI, which may be applied in detection and splitting of methylated DNA sections.

EFFECT: production of site-specific endonuclease PkrI.

3 dwg, 3 ex

Description

The invention relates to biotechnology and relates to the production of a new strain used to isolate a new site-specific endonuclease that recognizes and cleaves both chains of the methylated nucleotide sequence of 5′-GCN ↓ GC-3 ′ / 3′-CG ↓ NCG-5 ′ if her three or four 5-methylcytosines. An endonuclease with this specificity can be used for site-specific hydrolysis of DNA containing C5-methylcytosine bases.

To date, several site-specific endonucleases are known that recognize and cleave DNA, only if 5-methylcytosine is recognized in the site.

A known strain of Glacial ice bacterium I, producing GlaI endonuclease, which recognizes and cleaves the methylated nucleotide sequence 5′-G (m5C) ↓ GC-3 ′, where m5C is C5-methylcytosine [1].

A known bacterial strain Bacillus simplex 23, producing a site-specific BlsI endonuclease, cleaving the methylated nucleotide sequence 5′-GCNGC-3 ′ after the central nucleotide in the presence of two 5-methylcytosines nucleotide in this sequence [2].

The disadvantage of this strain is that the site-specific endonuclease produced by it cleaves all 5′-GCNGC-3 ′ sequences even if it contains at least two 5-methylcytosines, which does not allow it to be used for site-specific cleavage in DNA of only hypermethylated recognizable 5′-GCNGC-3 ′ sequences containing only three or four 5-methylcytosines.

A known strain of Bacillus subtilis T30 [3], producing a site-specific endonuclease BisI, which recognizes and cleaves the methylated nucleotide sequence of 5′-GCNGC-3 ′, in the presence of C5-methylcytosine bases in the recognition site.

A disadvantage of the known strain is that the BisI restriction endonuclease produced by it is not able to recognize and cleave both chains of the methylated nucleotide sequence of 5′-GC (m5C) GGC-3 ′ / 3′-CGG (m5C) CG-5 ′ DNA, where m5C is 5-methylcytosine.

Closest to the claimed strain, the prototype is the Glacial ice bacterium 24 strain producing the GluI restriction endonuclease, which recognizes the 5′-GCNGC-3 ′ nucleotide sequence and cleaves it in front of the central nucleotide (N) to form a single nucleotide 5′-protruding end, if all the cytosine bases of the recognizable sequence are methylated at position 5 (C5-methylcytosine) [4].

A disadvantage of the known strain is that the site-specific Glul endonuclease produced by it cannot cleave the recognizable 5′-GCNGC-3 ′ sequence after the central nucleotide, nor does it cleave this sequence when it contains three 5-methylcytosines.

An object of the invention is to obtain a bacterial strain producing a site-specific endonuclease that recognizes and cleaves both chains of the nucleotide sequence of 5′-GCNGC-3 ′ DNA in the presence of three or four 5-methylcytosines in it with the formation of a single nucleotide 3′-protruding end.

The technical task is achieved by obtaining a strain of Planomicrobium koreense 78k, a producer of site-specific endonuclease that recognizes and cleaves the methylated nucleotide sequence:

5′-G (m5C) N ↓ G (m5C) -3 ′

3 ′ - (m5C) G ↓ N (m5C) G-5 ′

where m5C is C5-methylcytosine. (The arrows indicate the positions of DNA cleavage.)

The proposed strain is isolated from natural material (soil) as a result of a targeted systematic search.

The obtained Planomicrobium koreense 78k strain was deposited in the All-Russian Collection of Industrial Microorganisms of the Federal State Unitary Enterprise State Research Institute for Genetics, and the site-specific endonuclease produced by it was named PkrI.

The strain Planomicrobium koreense 78k is characterized by the following features.

Cultural and morphological characters. Cells - gram-positive cocci, 1 μm in diameter, are located singly, in pairs, tetrads, motionless. When shaken in a broth, Luria form a homogeneous turbidity. On nutrient agar after 4 days at 24 ° C, colonies of about 1 mm form, smooth, shiny, opaque, pinkish in color.

Physiological and biochemical characteristics. Optional anaerobes. Catalopositive. Grow at a temperature of 4 to 40 ° C.

The content of G + C in DNA, equal to 55%, was determined by the method [5].

The strain was identified based on the analysis of morphological and biochemical properties according to the determinant [6], the site-specific endonuclease PkrI produced by the claimed strain was named according to the nomenclature [7].

The strain is stored in a freeze-dried state or in a solution of 30% glycerol at a temperature of -60 ° C.

For the cultivation of the strain, the following composition is used (g / l): peptone - 10, yeast extract - 5, NaCl - 5. Cultivation is carried out at 28 ° C with aeration until a stationary growth stage is reached.

The yield of the target enzyme is 100 u / g crude biomass with a concentration of 4000 u / ml.

The obtained site-specific endonuclease PkrI is characterized by the following properties:

1. Recognizes and cleaves the methylated nucleotide sequence 5′-GCN ↓ GC-3 ′ / 3′-CG ↓ NCG-5 ′ when there are three or four 5-methylcytosines in this sequence.

2. Does not cleave the above sequence without C5-methylcytosine bases (unmethylated) or less than three 5-methylcytosines.

3. Cleaves bonds after the central nucleotide in both chains of a recognizable sequence.

4. The optimal temperature of the enzyme is 30-37 ° C.

5. The optimal pH value for the action of the enzyme is 8.0-8.5.

6. The optimal salt concentration during DNA cleavage is 0-10 mM NaCl.

7. For the manifestation of RotI activity, Mg ²⁺ ions are required, the optimal concentration is 2-10 mm.

The defining difference between the proposed strain and the producer strains of site-specific endonucleases BlsI and GluI is that the first produces a site-specific endonuclease that recognizes and cleaves both chains of the methylated nucleotide sequence of 5′-GC ^∧ NGC-3 ′ DNA only under methylation conditions A recognizable sequence of more than three cytosines. This difference from Blsl allows the use of PkrI endonuclease for larger block fragmentation of C5 methylated DNA, and the difference from Glul for smaller block fragmentation of C5 methylated DNA.

Thus, the site-specific endonuclease PkrI is a new, unparalleled enzyme.

Since the proposed strain was obtained for the first time and was never used to isolate the site-specific endonuclease that recognizes and cleaves the above nucleotide sequence, we can conclude that the proposed strain meets the criteria of the invention of “novelty” and “inventive step”.

The invention is illustrated by the following examples of specific performance.

Example 1. Growing a strain and isolation of the enzyme.

To obtain biomass, the cells of the producer strain are transferred onto LB agar medium in a Petri dish and incubated overnight at 28 ° C. Freshly grown colonies are transferred with a sterile bacteriological loop to flasks containing LB liquid culture medium and cultivated on shakers at 28 ° C with stirring at 150 rpm until a stationary growth phase is reached. Cells are pelleted by centrifugation at 5000 rpm at 4 ° C. The biomass yield is 5 g / l of medium. Disintegration of cells, isolation and purification of the enzyme is carried out by a known method [8].

Example 2. Site-specific hydrolysis of C 5-methylated plasmid DNA with endonuclease PkrI.

DNA digestion was carried out under optimal conditions (37 ° C, reaction buffer 10 mM TrisHCl, pH 8.5, 10 mM MgCl ₂ , 100 mM NaCl, 1 mM DTT) for 60 minutes. Plasmid DNA cleavage products were separated by electrophoresis on a 1% agarose gel.

Various methylated and nonmethylated plasmid and phage DNAs are used as substrates for revealing the cleavage specificity: lambda and T7 phages, pHspAI (contains methylated sequences 5′-G (5mC) GC-3 ′ / 5′-G (5mC) GC-3 ′ [1]) and pFsp4HI2 (contains methylated sequences 5′-G (5mC) NGC-3 ′ / 5′-G (5mC) NGC-3 ′ [4]), as well as pUC19 DNA, pre-modified with CviPI methylase. CviPI methylase methylates cytosines at position C5 in the 5′-GC-3 ′ sequence [9].

The figure 1 presents the electrophoregram of products formed as a result of processing DNA phages of lambda and T7, as well as plasmids pHspAI and pFsp4HI2 by endonuclease PkrI.

Description of the tracks on the electrophoregram depicted in figure 1:

1 - DNA phage lambda;

2 - lambda phage DNA treated with PkrI endonuclease;

3 - DNA of phage T7;

4 - DNA of phage T7 treated with endonuclease PkrI;

5 - pHspAI DNA,

6 - pHspAI DNA treated with PkrI endonuclease;

7 - 1 kb DNA molecular weight marker (produced by NPO SibEnzyme);

8 - pFsp4HI2 DNA;

9 - pFsp4HI2 DNA treated with BlsI;

10 - pFsp4HI2 DNA treated with GluI;

11 - pFsp4HI2 DNA treated with PkrI.

As can be seen from figure 1, the endonuclease PkrI does not cleave unmethylated DNA phage lambda and T7, as well as pHspAI DNA containing methylated sequences 5′-GCGC-3 ′. At the same time, PkrI cleaves pFsp4HI2 DNA, giving a clearly visible fragment in the region of 450-500 bp. (lane 11). Theoretical calculations show that pFsp4HI2 DNA contains two 5′-GCNGCNGC-3 ′ sequences spaced 484 bp apart. The 5′-GCNGCNGC-3 ′ sequence contains two M.Fsp4HI methylase recognition sites that methylate the first cytosine in the sequence 5′-GCNGC-3 ′. Therefore, the 5′-GCNGC-3 ′ sequence in pFsp4HI2 should contain two 5-methylcytosines on both recognition site chains, and the 5′-GCNGCNGC-3 ′ sequence should contain three 5-methylcytosines: 5′-G (5mC) NG (5mC) NGC -3 ′ / 3′-CGN (5mC) GN (5mC) G-5 ′. This sequence includes two 5′-GCNGC-3 ′ sequences with three 5-methylcytosines. As theoretical calculations show, pFsp4HI2 has two sequences with three 5-methylcytosines (5′-G (5mC) NG (5mC) NGC-3 ′ / 3′-CGN (5mC) GN (5mC) G-5 ′) from each other at a distance of 485 bp, which corresponds to the electrophoretic mobility of the fragment formed during processing of pFsp4Ffl2 with PkrI endonuclease. This fact suggests that the endonuclease PkrI recognizes and cleaves the methylated 5′-GCNGC-3 ′ sequence containing three 5-methylcytosines. As can be seen in Figure 1, unlike PkrI, BlsI endonuclease effectively cleaves all methylated 5′-GCNGC-3 ′ sequences if it already has two 5-methylcytosines on both chains (lane 6). Unlike PkrI, endonuclease GluI does not cleave 5′-GCNGC-3 ′ sequences with either two or three 5-methylcytosines.

The figure 2 presents the electrophoregram of products formed by treating pUC19 DNA with Fsp4HI restriction endonuclease and pUC19 DNA pre-methylated with CviPI methylase and treated with PkrI endonuclease.

Description of the tracks on the electrophoregram depicted in figure 2:

1 - DNA19, hydrolyzed by MspI restriction endonuclease at the 5′-CCGG-3 ′ site, used as a molecular weight marker;

2 - DNA19U19 hydrolyzed by the restriction endonuclease Fsp4HI at the 5′-GCNGC-3 ′ site;

3 — pUC19 DNA pre-methylated with CviPI methylase and treated with PkrI endonuclease.

As can be seen from figure 2, the electrophoretic mobilities of the fragments formed during the hydrolysis of pUC19 according to the sequence of the recognition site of the restriction enzyme Fsp4HI and upon hydrolysis by the endonuclease PkrI of the plasmid pUC19, previously methylated by the sequence 5′-GC-3 ′, coincide.

This fact means that during C5 methylation of DNA according to the sequence 5′-GC-3 ′, the endonuclease PkrI cleaves the methylated sequences 5′-G (5mC) NG (5mC) -3 ′ / 3 ′ - (5mC) GN (5mC) G -5 ′ containing four 5-methylcytosines in a recognizable sequence.

Thus, the site-specific endonuclease PkrI recognizes and cleaves the 5′-GCNGC-3 ′ sequence only if it contains three or four 5-methylcytosines.

Example 3. Site-specific analysis and determination of the position of DNA hydrolysis by PkrI endonuclease on oligonucleotide duplexes.

The site of DNA hydrolysis by PkrI restriction enzyme was determined by comparing the lengths of the fragments formed upon cleavage by the restriction endonucleases PkrI and BlsI of the oligonucleotide duplex D1 / D2 formed from oligonucleotides D1 and D2:

D1: 5′-GAGTTTAG (m5C) GG (m5C) TATCGATCC-3 ′

D2: 5′-GGATCGATAG (m5C) CG (m5) CTAAACTC-3 ′.

As a marker of fragment lengths, products of partial cleavage of the same duplexes with exonuclease ExoIII were used.

Figure 3 shows a radio-autograph of an electrophoregram of the cleavage products of a deoxyribo-oligonucleotide radioactively labeled D1 / D2 duplex in a 20% polyacrylamide gel containing 7 molar urea.

Description of the tracks on the electrophoregram depicted in figure 3:

1 - source duplex D1 * / D2;

2 - D1 * / D2 duplex treated with PkrI endonuclease;

3 - duplex D1 * / D2 treated with exonuclease III from E. coli;

4 - D1 * / D2 duplex treated with BlsI endonuclease.

Oligonucleotides labeled with ³² P radioactive phosphorus at the 5′-end are indicated by *.

From figure 3 it is seen that the products of hydrolysis of DNA duplex D1 * / D2 in lanes 2 and 4 have the same lengths. This means that the positions of hydrolysis by the enzymes PkrI and BlsI coincide.

Based on the results obtained, it can be concluded that the endonuclease PkrI recognizes and site-specific cleaves the 5′-GCNGC-3 ′ DNA sequence after the central nucleotide in the presence of at least three 5-methylcytosines in this sequence.

The yield of site-specific PkrI endonuclease is determined by the electrophoretic picture of pFsp4HI2 DNA cleavage. The unit of activity is the minimum amount of enzyme required for complete cleavage of 1 μg of pFsp4HI2 DNA in 1 hour at a temperature of 37 ° C in a volume of 50 μl of the reaction mixture. The yield of the enzyme is 3000 u / g of raw biomass, the concentration of 4000 u / ml.

The enzyme is stored at -20 ° C in a buffer containing 50% glycerol, 0.2 M NaCl, 10 mM TrisHC1 (pH 7.5), 7 mM β-mercaptoethanol, 0.1 mM EDTA.

Thus, a new strain was obtained that produces a site-specific PkrI endonuclease that recognizes and cleaves both chains of the DNA nucleotide sequence, which contains three or four C5-methylcytosine bases at the 5'-GCNGC-3 'recognition site with the formation of a single nucleotide 5'-protruding end .

This site-specific endonuclease can be used in molecular biology, biotechnology and genetic engineering for site-specific cleavage of methylated DNA, in particular for the detection and analysis of methylated chromosomal DNA of mammals and humans.

INFORMATION SOURCES

1. Chernukhin V.A., Nayakshina T.N., Abdurashitov M.A., Tomilova Yu.E., Mezentseva N.V., Dedkov BC, Mikhnenkova N.A., Gonchar D.A., Degtyarev S. X. // New Glal restriction endonuclease recognizes the methylated sequence 5′-GCGC-3 ′. - Biotechnology. - 2006. - No. 4. - S.31-35.

2. Chernukhin V.A., Tomilova Yu.E., Chmuzh E.V., Sokolova O.O., Dedkov B.C., Degtyarev S.Kh. Site-specific endonuclease BlsI recognizes 5′-G (5mC) NGC-3 ′ DNA sequences and cleaves it to form 3′-protruding ends // Bulletin of Biotechnology and Physico-Chemical Biology named after Yu.A. Ovchinnikov. - 2007. - T.3. - No. 1. - S. 28-33.

3. Chmuzh E.V., Kashirina Yu.G., Tomilova Yu.E., Mezentseva N.V., Dedkov BC, Gonchar D.A., Abdurashitov M.A., Degtyarev S.Kh. New BisI restriction endonuclease from Bacillus subtilis T30 recognizes the methylated 5′G (m5C) ^∧ NGC-3 ′ DNA sequence // Biotechnology. - 2005. - No. 3. - S. 22-26.

4. Chernukhin V.A., Chmuzh E.V., Tomilova Yu.E., Nayakshina T.N., Gonchar D.A., Dedkov B.C., Degtyarev S.Kh. The new site-specific endonclease Glul recognizes the methylated DNA sequence 5′-G (5mC) NG (5mC) -3 ′ / 3 ′ - (5mC) GN (5mC) G-5 ′ // Journal of Biotechnology and Physicochemical Biology named after Yu .A. Ovchinnikova. - 2007. - T.3. - No. 2. - S.13-17.

5. Dedkov B.C. Determination of G + C content in bacterial DNA using restriction endonucleases // Biotechnology. - 2004. - No. 4. - S.77-82.

6. The determinant of bacteria Bergey. / Ed. J. Houlta et al. (9th edition in 2 volumes: Translated from English under the editorship of Acad. RAS G.A.Zavarzin). - M., 1997.

7. Smith H. O., Nathans D. A suggested nomenclature for bacterial host modification and restriction systems and their enzymes. // J. Mol. Biol. - 1973. - Vol. 81. - P. 419-423.

8. Bickle T.A., Pirrotta V. and Imber R. A simple, general procedure for purifying restriction endonucleases. // Nucleic Acids Res. - 1977. - V.4 - P.561-572.

9. Rebase official site http://rebase.neb.com/rebase/rebase.html

Claims (1)

The bacterial strain Planomicrobium koreense 78k, deposited under No. VKPM B-10627 in the All-Russian Collection of Industrial Microorganisms FSUE GosNIIIgenetika is the producer of the site-specific endonuclease PkrI, which recognizes and cleaves both chains of the DNA nucleotide sequence, which contains three or four C5-methylcytosine recognition bases 5 ′ -GCN ^∧ GC-3 ′ to form a single nucleotide 3′-protruding end.

Images