RU2340670C1 - Arthrobacter luteus B BACTERIA STRAIN-PRODUCER OF SITE-SPECIFIC Alu BI ENDONUCLEASE - Google Patents

Abstract

FIELD: chemistry; biotechnology.

SUBSTANCE: arthrobacter luteus B bacteria strain is obtained, which is a producer of site-specific Alu BI endonuclease. Restriction endonuclease recognises and splits both strands of the DNA 5'-AGCT-3' nucleotide sequence, without methylated bases and in such a case, when in the recognition site, one or two cytosines are methylated in the C5 position or one base is methylated in the N4 position.

EFFECT: new compounds with useful biological properties.

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Description

The invention relates to biotechnology and relates to the production of a new strain used to isolate a new restriction endonuclease that recognizes the nucleotide sequence of double-stranded DNA and cleaves it with the formation of blunt ends:

5'-AG ↓ CT-3 '

3'-TC ↑ GA-5 '.

Arrows indicate the positions of DNA cleavage.

An endonuclease with this specificity can be used for site-specific hydrolysis of DNA containing C5-methylcytosine bases, as well as when used with the AluI restrictase enzyme to identify and cleave methylated DNA regions of both prokaryotic and eukaryotic organisms, as well as phages and viruses.

Restriction endonucleases (restrictase, ER) are site-specific bacterial DNA endonucleases and, as a rule, are part of the so-called restriction-modification systems (PM systems). Currently, more than 250 different recognition sites for ER of the second type and more than 2000 different isothiisomers of restrictase enzymes, i.e. analogues of previously detected enzymes. Although the isoshizomers have the same recognition site with the original prototype, they may differ from the last in the place of DNA cleavage or in the ability to hydrolyze a recognizable sequence in the presence of methylated bases. The use of isoshizomers, differing in their ability to hydrolyze methylated DNA, has been widely used in studying the methylation status of natural DNA [1-4]. However, only a few examples are known of such pairs of isoschisomers, for example, EcoRII – MvaI [5, 6], or HpaII-MspI [7] used in research practice.

The closest to the claimed strain, the prototype, is the Arthrobacter luteus strain producing the AluI restrictase, which recognizes the 5'-AGCT-3 'nucleotide sequence and cleaves it before cytosine to form blunt ends [8].

A disadvantage of the known strain is that the site-specific endonuclease produced by it does not cleave a recognizable sequence if it contains at least one N4-methylcytosine or 5-methylcytosine [9].

An object of the invention is to obtain a bacterial strain producing a site-specific endonuclease that recognizes and cleaves both chains of the 5'-AGCT-3 'DNA nucleotide sequence, both without methylated bases, and if one or two cytosines are methylated at the recognition site in position C5 or one base in position N4.

The technical task is achieved by obtaining a strain of Arthrobacter luteus B-producer of restriction endonuclease Alu BI.

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

The resulting strain of Arthrobacter luteus B was deposited in the SibEnzyme microorganism culture collection under registration number 7M23, and the site-specific endonuclease produced by it was named Alu BI.

The strain Arthrobacter luteus B is characterized by the following features:

Cultural and morphological characters. On the medium, Luria-Bertrani (LB) forms white, smooth, shiny, opaque, convex, round colonies 4 mm in diameter. The cells are rod-shaped, 1x (3-5) microns in size, single, in pairs or in short chains. Form elliptical centrally located spores that do not inflate sporangia.

Physiological and biochemical characteristics. Gram-positive. Not capable of anaerobic growth, catalase-positive, oxidase-negative. They grow at a temperature of 10-40 ° C. The taxonomic affiliation of this strain is determined on the basis of the analysis of morphological and biochemical properties [10].

Based on the analysis of morphological and biochemical properties, the strain was identified as a species of the bacterium Arthrobacter luteus B, and the restriction endonuclease produced by it was named Alu BI according to the generally accepted nomenclature [11].

The restriction enzyme Alu BI is an isoschisomer of the restriction enzyme AluI, that is, it has the same hydrolysis position in the recognition site as AluI. Both of these enzymes do not cleave a recognizable sequence if it contains N6-methyladenine [12].

The main difference between the endonuclease produced by the claimed strain and the known restriction enzyme is that Alu BI, unlike AluI, is able to efficiently cleave double-stranded DNA when one or two cytosines at position C5 or one cytosine at position N4 is methylated at the recognition site.

The strain is stored in a lyophilically dried state or in a solution of 30% glycerol at a temperature of -20 ° C.

The resulting site-specific endonuclease Alu BI is characterized by the following properties:

1. Recognizes and cleaves the nucleotide sequence of 5'-AGCT-3 '.

2. Cleaves the bonds between guanine (G) and the cytosine base following it in both chains of a recognizable sequence.

3. Does not cleave a recognizable sequence containing one or two N6-methylcytosine bases, regardless of whether other methylated bases are present in this sequence or not.

4. Does not cleave a recognizable sequence containing two N4-methylcytosine bases.

5. Cleaves the above sequence containing one N4-methylated base and / or one or two C5-methylated base.

6. It is active in a wide temperature range of 20-50 ° C.

7. The optimal pH for the action of the enzyme is 7.5-8.5.

8. For the manifestation of Alu BI activity, Mg ²⁺ ions are required, the optimal concentration is 10 mM.

The defining difference between the proposed strain and the Arthrobacter luteus strain - producer of AluI ER is that the proposed strain produces a site-specific endonuclease Alu BI, which recognizes and cleaves both chains of the nucleotide sequence of 5'-AGCT-3 'DNA containing one or two C3-methylcytosine bases, whereas AluI is not able to hydrolyze the methylated sites in this way. Alu BI, unlike AluI, is able to hydrolyze the 5'-AGCT-3 'DNA sequence if one cytosine at position N4 is methylated in this double-stranded sequence.

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 examples of specific performance.

Example 1

Growing a strain and isolating an enzyme.

To obtain biomass, cells of the producer strain are transferred onto agarized LB medium in a Petri dish and incubated for 3 days at 22 ° C. Freshly grown colonies are transferred with a sterile bacteriological loop to flasks containing 300 ml of liquid LB nutrient medium and cultivated on shakers at 30 ° C with stirring at 150 rpm for two days until the 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.

The enzyme was isolated at 4 ° C by column chromatography.

37 g of frozen biomass was suspended in 100 ml of buffer A (10 mm Tris-HCl, pH 7.5, 0.1 mm EDTA, 7 mm β-mercaptoethanol) containing 0.05 M NaCl, 0.3 mg / ml lysozyme, 0.1 mM phenylmethylsulfonyl fluoride (PSMF), and incubated for 1 h with constant stirring. Then the cells were destroyed by ultrasound and centrifuged for 30 min at 15,000 rpm.

The supernatant was passed through a P-11 phosphocellulose column (Whatman, England) with a volume of 45 ml, previously equilibrated with buffer A containing 0.05 M NaCl, then a linear gradient of NaCl (0.05 M - 0.6 M) in buffer A 500 ml in volume, collecting 10 ml fractions. Fractions containing endonuclease were pooled, dialyzed against 20 volumes of buffer A, and loaded onto a column of 7 ml heparin-sepharose (Bio-Rad, USA). Elution was performed using a linear NaCl gradient (0.05 M - 0.5 M) in buffer A with a volume of 120 ml, collecting 3 ml fractions. The active fractions were combined and applied onto a 4 ml hydroxylapatite column, previously washed with equilibration buffer (0.01 M K ₂ HPO ₄ , pH 7.2, 7 mM β-mercaptoethanol). The column was washed with 10 ml of equilibration buffer and eluted by a linear gradient, collecting fractions of 2 ml each. Active fractions were combined and dialyzed against 20 volumes of concentration buffer (50% glycerol, 10 mM Tris-HCl, pH 7.55, 0.1 mM EDTA, 7 mM β-mercaptoethanol, 0.05 M NaCl). The drug was stored at -20 ° C.

The yield of the enzyme is 800 units of active enzyme with 1 g of biomass with specific activity of 5000 units / ml.

Example 2

Determination of the specificity of AluI restriction endonuclease.

The specificity of the enzyme is determined by the patterns of specific cleavage of various DNA (figure 1).

As substrates for revealing the specificity of cleavage, the phage λ and T7 DNA are used. After incubation under optimal conditions (37 ° C, SE-buffer "Y" - 33 mm Tris acetate, pH 7.9, 10 mm MgCl ₂ , 66 mm potassium acetate, 1 mm DTT) for 60 min at a concentration of plasmid DNA 0.02 mg / ml, reaction products are separated by electrophoresis on a 1% agarose gel.

Figure 1 presents the electrophoregram of the DNA cleavage products of phages λ and T7 restriction endonucleases AluI and Alu BI.

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

1 - DNA of phage λ;

2 - phage λ DNA treated with AluI ER;

3 - phage λ DNA treated with Alu BI ER;

4 - DNA of phage T7;

5 - phage T7 DNA treated with AluI ER;

6 - phage T7 DNA treated with Alu BI ER;

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

From figure 1 it is seen that when the DNA processing of phages λ and T7 by restriction endonucleases AluI and Alu BI as a result of DNA hydrolysis fragments of the same length are formed. These results suggest that AluI and Alu BI ERs recognize and cleave the same sequences on DNA.

Example 3

Determination of DNA cleavage sites by site-specific endonuclease Alu BI.

Determining positions DNA cleavage by site-specific endonuclease Alu BI was performed by comparing the lengths of the fragments formed by cleavage of labeled γ ^[32 P oligonucleotide duplexes Alu1 * / Alu2 and Alu2 * / Alu1, having a recognition sequence AluI endonucleases AluI and Alu BI (texte oligonucleotide structure duplexes and procedures for labeling oligonucleotides are shown in example 4). As a marker of fragment lengths, products of partial cleavage of the same duplexes with exonuclease ExoIII were used. The cleavage results of these oligonucleotide duplexes are shown in figure 2.

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

1 - duplex Alu1 * / Alu2;

2 - duplex Alu1 * / Alu2 cleaved by exonuclease ExoIII from E. coli;

3 - duplex Alu1 * / Alu2, split AluI;

4 - duplex Alu1 * / Alu2, split Alu BI;

5 - duplex Alu1 * / Alu2 cleaved by exonuclease III from E. coli;

6 - duplex Alu2 * / Alu1;

7 - duplex Alu2 * / Alu1 cleaved by exonuclease III from E. coli;

8 - duplex Alu2 * / Alu1, split AluI;

9 - duplex Alu2 * / Alu1, split Alu BI;

10 - duplex Alu2 * / Alu1, cleaved by exonuclease ExoIII from E. coli.

As can be seen from figure 2, the lengths of the fragments formed during the hydrolysis of oligonucleotide duplexes Alu1 * / Alu2 and Alu2 * / Alu1 are the same. Thus, the restriction endonuclease Alu BI cleaves the recognition site in the same position as AluI, that is, after guanine.

Example 4

Comparison of AluI and Alu BI ER methylation sensitivity.

The ability to cleave methylated ER recognition sites AluI and Alu BI was studied on synthetic oligonulotide duplexes containing methylated bases at the 5'-AGCT-3 'recognition site.

For experiments, restriction endonucleases AluI (concentration of 3000 units / μl), AluB I (concentration of 3000 units / μl), T4 polynucleotide kinase and buffer solutions manufactured by Sibenzyme (Russia) were taken. The oligodeoxyribonucleotides of the following composition, which served as a substrate for the endonucleases Alu I and AluB I, were synthesized in the Sibenzim NGO (Russia):

Alu1: 5'-GGT ATA GGA TGA AGCT TTC GCG GGT TAA GG-3 '

Alu2: 5'-SS TTA ACC CGC GAA AGCT TCA TCC TAT TSS-3 '

Alu3: 5'-GGT ATA GGA TGA (M6A) GCT TTC GCG GGT TAA GG-3 '

Alu4: 5'-SS TTA ACC CGC GAA (M6A) GCT TCA TCC TAT TCC-3 '

Alu5: 5'-GGT ATA GGA TGA AG (M5C) T TTC GCG GGT TAA GG-3 '

Alu6: 5'-SS TTA ACC CGC GAA AG (M5C) T TCA TCC TAT TCC-3 '

Alu7: 5'-GGT ATA GGA TGA AG (M4C) T TTC GCG GGT TAA GG-3 '

Alu8: 5'-SS TTA ACC CGC GAA AG (M4C) T TCA TCC TAT TSS-3 '

Oligonucleotides with an even number are complementary to oligonucleotides with an odd number. All oligonucleotide duplexes have the same primary structure and are distinguished from each other by the presence or absence of a methylated base in the AGCT sequence (the AGCT site recognized by AluI and Alu BI is underlined).

Preparation of substrate for AluI and Alu BI endonucleases.

One of the oligonucleotide duplex chains was modified at the 5'-end using T4 polynucleotide kinase and γ- [ ³² P] ATP. After the oligonucleotide was purified from reaction by-products, a complementary unlabeled oligonucleotide was added to it and the tube was heated for 2 minutes at 65 ° C, followed by cooling to room temperature on a worktable.

Hydrolysis of oligonucleotide duplexes by Alu I and AluB I. temperature 25 ° С), 10 mM Mg (СН ₃ СОО) ₂ , 66 mM KCH ₃ СОО, 1 mM DTT) and oligonucleotide duplex at a concentration of 66.7 nM at 37 ° С for 50 min.

The results of the cleavage of methylated and unmethylated synthetic oligonucleotide duplexes are shown in Fig.3-6.

Figure 3 shows the electrophoregram of the AluI and Alu BI restriction enzyme cleavage products of oligonucleotide duplexes containing the recognition site of these enzymes without methylated bases or one methylated base (semi-methylated site). As can be seen from figure 3, AluI and Alu BI effectively cleave oligonucleotide duplex containing unmethylated recognition site (lanes 2, 3). However, both enzymes are not efficiently able to cleave DNA if adenosine is methylated at least on one chain in the recognition site (lanes 4, 5). If only one DNA strand is methylated at the recognition site at position C5 or N4, then Alu BI cleaves the unmethylated strand (lanes 7, 9), while AluI does not cleave (lanes 6, 8).

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

1 - duplex Alu1 * / Alu2;

2 - duplex Alu1 * / Alu2, split AluI;

3 - duplex Alu1 * / Alu2, split Alu BI;

4 - duplex Alu1 * / Alu4 treated with AluI;

5 - duplex Alu1 * / Alu4 processed by Alu BI;

6 - duplex Alu1 * / Alu6 processed by AluI ;.

7 - duplex Alu1 * / Alu6, split Alu BI;

8 - duplex Alu1 * / Alu8 processed by AluI;

9 - duplex Alu1 * / Alu8, split Alu BI.

Figures 4 and 5 show electrophoregrams of the AluI and Alu BI enzyme cleavage products of oligonucleotide duplexes containing a recognition site for these enzymes with one or two methylated bases. As can be seen from FIG. 4, Alu BI cleaves the methylated chain at the recognition site if one (lane 3) or two (lane 5) C5-methylcytosine bases is included in the recognition sequence; however, AluI cannot hydrolyze these methylated DNA chains (lanes 2, 4). Thus, Alu BI, unlike AluI, hydrolyzes both DNA strands at the recognition site if it contains one or two C5-methylcytosine bases. However, the C5 methylated chain is not hydrolyzed by AluI and Alu BI if the second DNA chain contains an N6 methylated base (lanes 6,7). Alu BI, unlike AluI, hydrolyzes the C5 methylated chain in the presence of an N4 methylated base on the opposite chain (lanes 8, 9).

Description of the tracks on the electrophoregram depicted in figure 4:

1 - duplex Alu5 * / Alu2;

2 - duplex Alu5 * / Alu2 treated with AluI;

3 - duplex Alu5 * / Alu2, split Alu BI;

4 - duplex Alu5 * / Alu6 treated with AluI;

5 - duplex Alu5 * / Alu6, split AluI;

6 - duplex Alu5 * / Alu4 processed by Alu BI;

7 - duplex Alu5 * / Alu4 treated with AluI;

8 - duplex Alu5 * / Alu8 processed by AluI;

9 - duplex Alu5 * / Alu8, split Alu BI.

As can be seen from figure 5, Alu BI hydrolyzes the N4-methylated chain if the complementary chain does not contain methylated bases in a recognizable sequence (lane 3), while AluI is not able to hydrolyze this chain (lane 2). However, both AluI and Alu BI do not hydrolyze the recognition site if it contains two N4-methylated bases (lanes 4, 5). Thus, Alu BI, unlike AluI, can hydrolyze both DNA strands if one N4-methylated base is included in the recognizable sequence. An N4-methylated chain can also hydrolyze if the complementary chain of the recognized sequence also contains one methylated base -5m-cytosine (lane 7), while AluI is not able to hydrolyze this chain (lane 6). Thus, if there are two methylated bases in the recognition site, Alu BI hydrolyzes both DNA chains on one chain of the C5 methylated base and the other on the N4 methylated base, while AluI does not hydrolyze either of the chains. A DNA chain containing a single methylated base, N4-methylcytosine in a recognizable sequence, will not be hydrolyzed by either Alu BI or AluI if 6m-adenine is contained in the complementary chain in a recognizable sequence (lanes 8, 9).

Description of the tracks on the electrophoregram depicted in figure 5:

1 - duplex Alu7 * / Alu2;

2 - duplex Alu7 * / Alu2 processed by AluI;

3 - duplex Alu7 * / Alu2, split Alu BI;

4 - duplex Alu7 * / Alu8 processed by AluI;

5 - duplex Alu7 * / Alu8 processed by Alu BI;

6 - duplex Alu7 * / Alu6 processed by AluI;

7 - duplex Alu7 * / Alu6, split Alu BI;

8 - duplex Alu7 * / Alu4 processed by AluI;

9 - duplex Alu7 * / Alu4 processed by Alu BI.

Example 5

Use of Alu BI ER to determine the methylation status of bacterial chromosomal DNAs and for the selective hydrolysis of C5-methylated DNA.

Due to the different sensitivity to cytosine methylation in both C5 and N4 positions, Alu BI together with AluI can be used to determine the presence or absence of C5 and N4-methylated bases in the 5'-AGCT-3 'sequence in both bacterial and and eukaryotic viral and genomic DNA.

Figure 6 shows the electrophoregram of the chromosomal DNA of Arthrobacter luteus, a producer of AluI ER, treated with restriction enzymes AluI and Alu BI.

The chromosomal DNA of the Arthrobacter luteus producer AluI strain is known to contain 5-methylcytosine at the 5'-AGCT-3 'site [13]. In accordance with the properties of the enzymes Alul and Alu BI described above, the chromosomal DNA of Arthrobacter luteus, the producer of AluI, is not hydrolyzed by AluI restrictase but is efficiently cleaved by Alu BI restrictase.

Description of the tracks on the electrophoregram depicted in Fig.6:

1 - chromosomal DNA of Arthrobacter luteus - producer AluI;

2 - chromosomal DNA of Arthrobacter luteus - AluI producer, treated with AluI restrictase;

3 - chromosomal DNA of Arthrobacter luteus - producer of AluI, treated with restriction enzyme Alu BI;

4 - 1 kb DNA molecular weight marker (produced by NPO SibEnzyme).

Thus, a new strain was obtained that produces the Alu BI restriction endonuclease, which is capable of efficiently cleaving double-stranded DNA when one or two cytosines at position C5 or one cytosine at position N4 is methylated at the recognition site.

Claims (1)

The bacterial strain Arthrobacter luteus B, deposited in KKM NPO SibEnzyme under the number 7M23 is the producer of the site-specific endonuclease Alu BI.

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