Valery A. Chernukhin, Vladimir S. Dedkov, Danila A. Gonchar, Murat A. Abdurashitov, Alexander G. Akishev, Tatyana N. Nayakshina, Elena N. Lomakovskaya and Sergey Kh. Degtyarev*
SibEnzyme Ltd., Novosibirsk, 630117, Russia
* the Author for correspondence: E-mail: email@example.com
A cloning of GlaI site-specific methyl-directed DNA endonuclease gene and the comparison of the substrate specificity of recombinant and native enzymes is described. The analysis of recombinant GlaI endonuclease properties showed that the substrate specificity of native and recombinant enzymes didn’t differ, but the concentration of recombinant enzyme exceeded 10 times of native enzyme.
Abbreviations: 5mC – 5-methylcytosine, BSA – bovin serum albumin, DMSO – dimethylsulfoxide, DTT – dithiothreitol, EDTA – ethylenediaminetetraacetic acid, IPTG – Isopropyl-ß-D-1-thiogalactopyranoside, MD endonuclease – methyl-directed DNA endonuclease, MTase – DNA Methyltransferase, N – any nucleotide, PAAG – polyacrylamide gel, Tris – tris-(oxymethyl)-aminomethane
The site-specific methyl-directed DNA endonucleases, or MD endonucleases – are enzymes which recognize and cleave methylated DNA sequences only and don’t hydrolyze unmodified DNA . These enzymes are similar to well-studied restriction endonucleases because they don’t require any cofactors with the exception of Mg2+ ions for its activity. GlaI MD endonuclease isolated from the bacterial strain Glacial ice bacterium recognizes the DNA sequence 5’-R(5mC)GY-3’ (where R – purine, Y – pyrimidine) in the presence of 5-methylcytosine in a complementary chain, and cleaves both chains of DNA in the middle of the site with formation of blunt ends . It is noteworthy that mammalian DNMT3 DNA Methyltransferase modifies DNA de novo with formation of the sequence 5’-R(5mC)GY-3’ . So, GlaI found application in epigenetic DNA diagnostics, e.g. it was successfully used for determination of the methylation status of the local sites or fragments of DNA [4,5] and for establishment of the human epigenome as well . Since GlaI application in the epigenetic DNA diagnostics is effective provided when the complete specific DNA digestion of the analyzed DNA samples is achieved, the high enzyme concentration being necessary. However, a large amount of Glacial ice bacterium strain biomass can only partially solve the problem of the enzyme production with high concentration. Cloning of GlaI gene in E.coli allows to significantly increase the enzyme concentration in biomass and to receive highly active recombinant enzyme from rather small amount of the bacterial cells.
The site-specific methyl-directed DNA endonucleases are new and poorly studied enzymes. In this work we have undertaken a comparison of substrate specificity of the native and recombinant enzymes.
MATERIALS AND METHODS
Reagents from following manufacturers were used in this study: “Sigma-Aldrich” (USA), “Fisher” (USA), “Panreac” (Spain) and “Helicon” (Russia). The following resins were used for chromatographic purification of enzyme: Phosphocellulose P-11 (“Whatman”, England), Heparin-sepharose (“Bio-Rad”, USA), Hydroxyapatite (“BioRad”, USA). The strain was grown using the medium components made by ”Organotechnie” (France).
The Esherichia coli ER2267 strain (F’ proA B lacIq Δ(lacZ) M15 zzf:: mini-Tn10 (Kanr)/Δ (argF-lacZ)U169 glnV44 e14 (McrA-) rfbD1. recA1 relA1. endA1 spoT1. Thi-1 Δ(mcrC-mrr) 114:: IS10) was from “New England Biolabs, Inc.” (USA). For experiments we used DNA of λ bacteriophage and a variety of C5-methylated plasmids which were constructed before [7-9]. We used 1 kb DNA Ladder (“SibEnzyme”, Russia) as a marker of the molecular weight of DNA fragments. For GlaI enzyme preparation dilution the SE Buffer “B100” (10 mM Tris-HCl, pH 7.6, 50 mM KCl, 0.1 mM EDTA, 200 μg/ml BSA, 1 mM DTT, 50% glycerol) (“Sibenzyme”, Russia) was used. All experiments on the activity assay and substrate specificity determination of recombinant and native GlaI were carried out in 20 μl of the reaction mixture containing the SE Buffer “Y” (33 mM Tris-acetate, pH 7.9, 10 mM Mg-acetate, 66 mM K-acetate, 1 mM DTT) and 0.5 μg of substrate DNA at 30°C for 1 hour.
GlaI gene cloning
The DNA of Glacial ice bacterium strain was isolated as described earlier , and it was partially digested with Kzo9I restriction endonuclease (5’-GATC-3’). The Kzo9I-fragments were ligated in pUC19 vector  previously linearized by BamHI restriction endonuclease, and, then, the library of the recombinant clones E.coli ER2267 carrying plasmids with Glacial ice bacterium genome DNA fragments was obtained by a standard technique . The clones were analyzed on the presence of GlaI activity in a bacterial cell lysate using pHspAI plasmid DNA  previously linearized by DriI restriction endonuclease (pHspAI/DriI) by the method described earlier .
Growing cells of E.coli ER2267 (pMTL22-GlaI) recombinant strain carrying the gene of GlaI
For recombinant GlaI biomass obtaining a single colony of the E.coli ER2267 (pMTL22-GlaI) strain was sowed in 0.5-liter flasks with 100 ml of LB medium (1% tryptone, 0.5% yeast extract, 0.5% NaCl, рН 7.5) with addition of ampicillin (50 μg/ml). The culture was grown overnight in the thermostat at 37°C. Then the inoculum was sowed to 12 flasks (5 ml inoculum to each flask) with 100 ml of LB medium containing ampicillin. The culture grew in thermoshaker within 16 hours at 30°C with stirring at 140 rpm. After 11 hours of cultivation the IPTG was added into the culture to 1 mM, after that it grew more 5 hours. Then 1 ml of cultural liquid was picked out for the analysis, and the cells were collected by centrifugation for 30 minutes at 3000 g on the Beckman J2-21 centrifuge (Beckman, USA) and were frozen at -20°C.
For the analysis of target activity, the cells from 1 ml of cultural liquid were precipitated by centrifugation on the Eppendorf centrifuge at 12000 rpm for 2 min, and were suspended in 100 μl of the Lysis-buffer (10 mM Tris-HCl, pH 8.5, 0.1 mg/ml Lysozyme, 50 mM NaCl, 1 mM EDTA, 0.1% Triton X-100) for 30 min using “Multi-vortex V-32” (“Biosan”, Lithuania). 8 μl of crude lysate was added to 40 μl of the reaction mixture containing 1x SE buffer “Y” and 0.5 μg of pHspAI/DriI plasmid DNA which was used earlier for native GlaI activity assay  in the microplate well. The suspension was mixed by pipetting and 20 μl of this one was transferred to the following well with 20 μl of the same reaction mixture to make the dilution of the lysate in 2 time. Such procedure was repeated to 7 passage. Reaction mixture was incubated for 1 hour at 30°C, and the reaction was stopped by an addition of the Stop-buffer (40% sucrose, 0.1 M EDTA, 0.05% bromophenol blue).
Isolation of recombionant GlaI enzyme from biomass
All procedures of enzyme isolation were performed at 4°C. 40 g of frozen cells of E.coli ER2267 (pMTL22-GlaI) were suspended in 30 ml of Buffer A (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 7 mM β-mercaptoethanol) containing 0.3 M NaCl, 0.3 mg/ml Lysozyme and 0.1 mM phenylmethylsulphonyl fluoride (PMSF). Biomass was lysed in a glass flask with stirring on a magnetic stirrer for 1 hour. As a result of cells lysis the solution became more viscous and transparent. The obtained lysate was disrupted by ultrasonic disintegrator Soniprep 150 (MSE, England) with an adapter diameter of 2 cm, 5 times of 30 seconds with intervals in 1 min for cooling of suspension in an ice bath. Further the lysate was clarified by centrifugation on J2-21 centrifuge (“Beckman”, USA) at 15000 rpm for 30 min.
The supernatant was initially applied to a 20 ml of Phosphocellulose P-11 column pre-equilibrated with Buffer A containing 0.1 M NaCl, and washed with two column volumes of the same buffer. Enzyme elution was performed with 200 ml of a linear gradient of NaCl concentration (0.2-0.65 M) in Buffer A. 20 fractions of 10 ml were collected. Endonuclease-containing fractions were pooled and dialyzed against 20 volumes of Buffer A with 0.1 M NaCl and applied on a column with 10 ml of Heparin-sepharose. Enzyme elution was carried out with 200 ml of a linear gradient of NaCl (0.1-0.45 M) in Buffer A. 50 fractions of 4 ml were collected. Target fractions were pooled and applied on the column with 4 ml of Hydroxyapatite pre-equilibrated with Buffer B (10 mM K-phosphate, pH 7.2, 0.1 mM EDTA, 7 mM β-mercaptoethanol) containing 0.02 M NaCl. The column was washed with 10 ml of the same buffer, and target protein was eluted by a linear gradient of K-phosphate buffer, pH 7.2, from 0.01 to 0.4 M K-phosphate with a volume of 100 ml. 40 fractions of 2.5 ml were collected. Active fractions were pooled, dialyzed against 20 volumes of the concentrating buffer (10 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 0.1 mM EDTA, 0.05% Triton X-100, 7 mM β-mercaptoethanol, 50% glycerol). The enzyme preparation was stored at -20°C.
RESULTS AND DISCUSSION
As a result of determination of GlaI MD endonuclease activity in lysates of E.coli clones from pUC19/BamHI-Gla DNA/Kzo9I library with use of pHspAI/DriI DNA-substrate the recombinant plasmid with a target gene of GlaI was isolated. Then the PCR fragment including target gene was ligated into the FauNDI-BamHI sites of pMTL22 . E.coli ER2267 cells were transformed by the obtained construction, pMTL22-GlaI, and the recombinant strain E.coli ER2267 (pMTL22-GlaI) was selected and used further for biomass growing and subsequent enzyme isolation. Results of testing of target activity in a lysate of cells of the recombinant E.coli ER2267 (pMTL22-GlaI) biomass are given in figure 1.
Figure 1. The target activity analysis in biomass of the recombinant strain-producer – E.coli ER2267 (pMTL22-GlaI), on pHspAI/DriI plasmid DNA.
Lanes: 1-8 – serial twice lysate dilution of the biomass, since 4 μl of lysate; M – 1 kb molecular weight DNA ladder (from 0,25 to 10 kb). Electrophoresis in 1% agarose gel.
As can be seen from Figure 1, the complete hydrolysis of the substrate DNA is observed on lane 1, and the picture of hydrolysis corresponds to a fragments set, which is typical for native GlaI .
As a result of GlaI MD endonuclease isolation by chromatographic purification 6 ml of enzyme preparation with activity of 100.000 units/ml was obtained from 10 g of the recombinant strain-producer, that was 10 times higher than the activity of GlaI isolated from the native strain (up to 10.000 units/ml).
Analysis of substrate specificity of recombinant GlaI.
As was shown earlier for native GlaI it’s activity increased at 20-30 times in the presence of 20% DMSO . Therefore, comparison of substrate specificity of recombinant and native GlaI on pHspAI/DriI DNA in the presence of different concentration of DMSO was carried out. The reactions of DNA digestion were carried out in SE buffer “Y” in the absence or the presence of DMSO (10%, 20%, 25%) at optimal temperature (30°C) for 1 hour.
In the Figure 2 a comparison of activities of GlaI preparations isolated from native and recombinant strains is presented. For the convenience of the activities comparison the recombinant GlaI preparation was diluted in 4 times with SE Buffer for enzymes storage and dilution “B100”.
Figure 2. Comparison of substrate specificity of GlaI isolated from the native (I) and recombinant (II) bacterial strains on pHspAI/DriI DNA. Electrophoresis in 12% PAAG. On the right theoretically calculated picture of pHspAI/DriI plasmid digestion on the sites 5’-R(5mC)GY-3’ is given.
Lanes: 1-6 – serial twice enzyme preparation dilution, since 1 μl of the preparations; M – pUC19/MspI molecular weight DNA ladder.
From the Figure 2 it is visible that recombinant GlaI, like native enzyme, is activated by presence in the reaction mixture of DMSO, and the greatest activity is shown at the DMSO concentration of 20%. This figure also showed that the substrate specificity of the both enzyme preparations on pHspAI/DriI doesn’t differ, however DNA digested with the recombinant GlaI preparation, even diluted by 4 times, revealed more complete digestion of the DNA-substrate in comparison with the native GlaI preparation.
Further we carried out the comparative analysis of activities of native and recombinant GlaI preparations on various methylated DNA substrates. As substrates we used phage λ DNA and the following plasmid DNA:
– pMHpaII (including the gene of HpaII DNA MTase modifying the second cytosine in the 5 ‘-CCGG-3’ sequence, and containing the 5’-C(5mC)GG-3’/3’-GG(5mC)C-5’ sites );
– pMHaeIII (including the gene of HaeIII DNA MTase modifying the first cytosine in the 5 ‘-GGCC-3′ sequence, and containing the 5’-GG(5mC)C-3’/3’-C(5mC)GG-5’ sites );
– pFsp4HI3 (including the gene of Fsp4HI DNA MTase modifying the first cytosine in the 5′-GCNGC-3′ sequence, and containing the 5’-G(5mC)NGC-3’/3’-CGN(5mC)G-5’ sites );
– pBspACI (including the genes of BspACI-1 and BspACI-2 DNA MTases modifying the cytosines in the 5′-CCGC-3′ and 5′-GCGG-3′ sequences, and containing the 5’- (5mC)CGC-3’/3’-GG(5mC)G-5’ sites );
– pHspAI (including the gene of HspAI DNA MTase modifying the first cytosine in the 5′-GCGC-3′ sequence, and containing the 5’-G(5mC)GC-3’/3’-CG(5mC)G-5’ sites ).
For the convenient to the DNA hydrolysis analysis all plasmid DNAs were linearized before with DriI restriction endonuclease. Reactions were carried out in the SE Buffer “Y” in the presence of 20% DMSO.
The results of cleavage of these substrates with recombinant GlaI are presented in the Figure 3.
igure 3. Analysis of substrate specificity of recombinant GlaI. Electrophoresis in 1% agarose gel. Lanes: I – Lambda; II – pMHpaII/DriI; III – pMHaeIII/DriI; IV-pFspAI3/DriI; V – pBspACI/DriI; VI – pHspAI/DriI; 1 – DNA without enzyme addition; 2 – DNA with addition of recombinant GlaI; 3 – DNA with addition of native GlaI; M – 1 kb molecular weight DNA ladder (from 0,25 to 10 kb).
As can be seen from this figure recombinant GlaI like the native enzyme doesn’t cleave unmethylated DNA substrate (Lambda) and all C5-methylated substrates as well, except pHspAI plasmid. This fact confirms that recombinant GlaI, as well as the native one, recognizes and cuts only the sequence 5’-R(5mC)GY-3’ containing the 5-methylcytosine.
The received results confirm that GlaI isolated from the recombinant strain, having 10 times bigger activity, and reveals the same substrate specificity, as the enzyme from a native (Glacial ice bacterium) strain. As a result it is possible to make the conclusion that use of recombinant GlaI allows to achieve more complete DNA samples digestion that will undoubtedly improve the results of epigenetic researches with the use of this enzyme.
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