Translated from "Ovchinnikov bulletin of biotechnology and physical and chemical biology" V.7, No 3, pp 35-42, 2011
We have discovered and purified a new methyl-directed site-specific DNA endonuclease PkrI from bacterial strain Planomicrobium koreense 78k. PkrI recognizes and cuts methylated DNA sequence 5'-GCNGC-3'/3'-CGNCG-5' carrying at least three 5-methylcytosimes and doesn’t cleave unmethylated DNA. Due to its ability to cleave only modified DNA PkrI may find a practical application in genetic engineering experiments as well as in determination of DNA methylation status.
Type II restriction endonucleases are the most known and well studied enzymes among all site-specific DNA endonucleases. As a rule restriction endonuclease (RE) and corresponding DNA-methyltransferase form restriction-modification system (RM-system). RE cleaves foreign DNA at a short recognition site, whereas a cognate MTase modifies the same sequence in a host DNA protecting it against digestion with RE.
Methyl-directed site-specific endonucleases (MD endonucleases) hydrolyze only methylated DNA and their biochemical properties are similar to the restriction endonucleases ones. Recently discovered at SibEnzyme site-specific 5-methylcytosine directed DNA endonucleases recognize and cleave different methylated DNA sequences, require only Mg2+ ions as a cofactor and completely hydrolyze DNA .
Three MD endonucleases BlsI , BisI  and GluI  recognize different variants of methylated 5'-GCNGC-3’ sequence and cleave DNA before or after N.
In the present work we describe a substrate specificity of new methyl-directed DNA-endonuclease PkrI, which recognizes methylated DNA sequence 5’-GCN↑GC-3’/3’-CG↓NCG-5’carrying at least three 5-methylcytosines and cleaves it as indicated by arrows.
The strain producer growth. The strain was grown in the fermenter 1601-013 (LKB, Sweden) at 30°C in 10 L of nutrient medium containing 1% Tryptone (Organotechnie, France), 0.5% yeast extract (Organotechnie, France), 0.5% NaCl, and 0.05% MgCl2, pH 7.5, with aeration at 10 L/min and stirring at 200 rev/min. At a late logarithmic stage of growth the bacterial cells were precipitated by centrifugation. Pellet of cells (100 g) was stored at -20°C.
Enzyme isolation. All procedures of enzyme isolation were performed at 4°C. The following buffer solutions were used:
Buffer A - 10 mM Tris-HCl, pH 7.6, 0.1 mM EDTA and 7 mM β-mercaptoethanol;
Buffer B - 10 mM K-phosphate, pH 7.2, 0.1 mM EDTA and 7 mM β-mercaptoethanol;
Buffer C- 60 mM Tris-HCl, pH 7.6, 0.1 mM EDTA and 7 mM β-mercaptoethanol.
15 grams of frozen cells were suspended in 45 ml of the Buffer A, containing 0.3 M NaCl, 0.1 mg/ml of lysozyme, 1 mM phenyl-methyl-sulphonyl fluoride (PMSF) and 0.1% triton X-100 and mixed with a stirrer during 1 hour at 4°C. The cells were disrupted by ultrasonic disintegrator Soniprep 150 (MSE, England) 7 times for 1 min with 1 min intervals to cool the suspension.
The crude lysate was clarified by centrifugation for 30 min at 15.000 rpm in JA-20 rotor (J2-21 centrifuge, Beckman, USA).
Phosphocellulose chromatography. The supernatant was initially applied to a P-11 phosphocellulose column (45 ml) pre-equilibrated with Buffer A containing 0.2 M NaCl. The column was washed with 2 volumes of the Buffer A with 0.2 M NaCl. A flow through fraction with PkrI activity was precipitated with 70% ammonium sulphate with subsequent centrifugation for 30 min at 12.000 rpm in JA-14 rotor (J2-21 centrifuge).
Gel filtration. The precipitate was diluted with the Buffer A and loaded onto the Biogel A-0.5m column (500 ml), washed with the Buffer A containing 0.8 M NaCl and 0.1% Triton X-100.
DEAE-cellulose chromatography. Endonuclease-containing fractions were combined and dialyzed against buffer A during 3 h and were applied to the DEAE-cellulose column (50 ml) pre-equilibrated with buffer A containing 0.05 M NaCl. The column was washed with 2 volumes of Buffer A containing 0.05 NaCl. The enzyme was eluted with 400 ml of a linear gradient of the NaCl concentration (0.075 M-0.45 M) in Buffer A.
Phenyl-sepharose chromatography. Endonuclease-containing fractions were combined, adjusted to 60 mM Tris-HCl, pH 7.6 and sulphate ammonium was added to a final concentration 1.7 M. The obtained solution was applied on the Phenyl-sepharose column (4 ml) pre-equilibrated with Buffer C containing 1.7 M ammonium sulphate and washed with 2 volumes of the same Buffer. The enzyme was eluted with 240 ml of a linear gradient of the ammonium sulphate (1.7 M-0 M) in Buffer C.
Hydroxyapatite chromatography. Endonuclease-containing fractions were combined and dialyzed against 2.5 l of the Buffer A during 16 h, then were applied to the Hydroxyapatite column (4 ml) pre-equilibrated with Buffer B containing 0.01 % Triton X-100. The column was washed with 2 volumes of the Buffer B, and the protein was eluted with 160 ml of a linear gradient of the K-phosphate Buffer (0.01 M-0.2 M).
Phenyl-sepharose rechromatography. Endonuclease-containing fractions were combined, adjusted to 60 mM Tris-HCl, pH 7.6 and sulphate ammonium was added to a final concentration 1.7 M. The obtained solution was applied on the Phenyl-sepharose column (4 ml) pre-equilibrated with Buffer C containing 1.7 M ammonium sulphate and washed with 2 volumes of the same Buffer. The enzyme was eluted with 200 ml of a linear gradient of the ammonium sulphate (1.7 M-0 M) in Buffer C.
Heparin-sepharose chromatography. Endonuclease-containing fractions were combined and dialyzed against 1.5 l of the Buffer A during 16 h, then were applied to the Heparin-sepharose column (4 ml) pre-equilibrated with Buffer A containing 0.1 M NaCl and washed with 2 volumes of the same Buffer. The enzyme was eluted with 160 ml of a linear gradient of the NaCl concentration (0.1 M-0.7 M) in Buffer A.
Enzyme concentrating and storage. Fractions with a maximal enzyme’s activity were pooled and concentrated by dialysis against 300 ml of Buffer A with 50% glycerol, 0.25 M NaCl during 20 h. Purified enzyme was stored at -20°C.
Enzyme activity determination. The plasmid pFsp4HI2 was used as a substrate for determination of PkrI activity. PkrI activity was determined in 20 μl of a reaction mixture containing 0.5 μg pFsp4HI2/DriI  in SE-buffer “B” for 1 hour at 37°C. One unit of PkrI activity is defined as the amount of enzyme required to completely digest 1 μg of pFsp4HI2/DriI DNA in SE Buffer “B” at 37°C (optimal conditions) for 1 hour in a total reaction volume of 50 μl.
Determination of PkrI recognition site and cleavage position on oligonucleotide duplexes. The position of DNA hydrolysis with PkrI was determined by comparison of DNA fragments lengths after cleavage of [γ32P]-labeled synthetic oligonucleotide duplex D1/D2 with PkrI and BlsI endonucleases. A partial cleavage product of one of this duplex by Exonuclease III from E.coli (ExoIII) was used as a fragment length marker.
The digestion has been performed at optimal conditions for 1 hour. The products of hydrolysis were separated by electrophoresis in denaturating 20% PAAG with 7 M Urea in TBE Buffer.
Oligodeoxyribonucleotides were synthesized at SibEnzyme Ltd. (Russia). The duplexes from the following oligonucleotides were used as the substrates for PkrI endonuclease:
NN02 5’- GCTTGTACTTTAGCGGCATTGATTCTCACCACG-3’
Oligonucleotides with even number are complementary to oligonucleotides with odd number.
Determination of the primary structure of 16S ribosomal RNA gene has been performed using PCR of strain-producer chromosomal DNA with the following primers:
16S ribosomal RNA gene primary structure was determined by the 3130xl Genetic Analyzer (Applied Biosystems).
Strain-producer description and its taxonomical identification. The strain-producer of PkrI endonuclease was isolated from the soil sample. The bacterial cells are gram-positive immobile cocci with a diameter of 1 μm. The cells are arrange separately or aggregated in pairs or tetrads. Catalase-positive and facultative aerobic. They grow at the temperature of +4-40°. The bacterial culture in LB forms homogeneous turbidness after shaking. It forms pinkish, smooth, bright, non-transparent colonies 1 mm in diameter after 4 day cultivation on Luria-Betrani medium agar. GC-content 52-58% was determined according to the previously described method .
16S gene primary structure is presented below:
1 ccatcatctg tccaaccttc ggcggctggc tccacaaggg ttacctcacc gacttcgggt
61 gttacaaact ctcgtggtgt gacgggcggt gtgtacaagg cccgggaacg tattcaccgt
121 ggcatgctga tccacgatta ctagcgattc cggcttcatg caggcgagtt gcagcctgca
181 atccgaactg agaacggttt tctgggattg gctccccctc gcgggtttgc agccctttgt
241 accgtccatt gtagcacgtg tgtagcccag gtcataaggg gcatgatgat ttgacgtcat
301 ccccaccttc ctccggtttg tcaccggcag tcaccttaga gtgcccaact gaatgctggc
361 aactaagatc aagggttgcg ctcgttgcgg gacttaaccc aacatctcac gacacgagct
421 gacgacaacc atgcaccacc tgtcaccgct gtccccgaag ggaaagccta gtctcctagg
481 cgggcagcgg gatgtcaaga cctggtaagg ttcttcgcgt tgcttcgaat taaaccacat
541 gctccaccgc ttgtgcgggc ccccgtcaat tcctttgagt ttcagccttg cggccgtact
601 ccccaggcgg agtgcttaat gcgttagctg cagcactaag gggcggaaac cccctaacac
661 ttagcactca tcgtttacgg cgtggactac cagggtatct aatcctgttt gctccccacg
721 ctttcgcgcc tcagcgtcag ttacagacca gaaagtcgcc ttcgccactg gtgttcctcc
781 acatctctac gcatttcacc gctacacgtg gaattccact ttcctcttct gcactcaagt
841 cccccagttt ccaatgaccc tccaggttga gccgtgggct ttcacatcag acttaaggga
901 ccgcctgcgc gcgctttaca cccaataatt ccggacaacg cttggcacct
Based on morphological and biochemical properties  and nucleotide sequence of 16S ribosomal RNA gene , the bacterial strain was identified as Planomicrobium koreense 78k. DNA endonuclease was named PkrI according to the standard nomenclature .
Enzyme purification and determination of PkrI substrate specificity. PkrI was purified from the cell extract by consecutive chromatographic steps as described in “Materials and Methods”. Data on PkrI activity determination are given in Figure 1. The concentration of PkrI was determined to be 2000 units/ml. A total volume of PkrI preparation was 3 ml.
Figure 1. Determination of PkrI activity in hydrolysis of pFsp4HI2/DriI DNA. Electrophoresis in 1% agarose gel. Lanes:
1 – 2 μl PkrI preparation; 2 – 1 μl PkrI preparation;
3 – 1/2 μl PkrI preparation; 4 – 1/4 μl PkrI preparation;
5 – 1/8 μl PkrI preparation; 6 – 1/16 μl PkrI preparation;
7 – 1/32 μl PkrI preparation; 8 – 1/64 μl PkrI preparation;
9 – DNA without PkrI preparation; 10 – 1 kb DNA ladder (“SibEnzyme”, Russia).
Substrate specificity of PkrI has been determined by hydrolysis of different plasmid and phage DNAs: lambda and T7 phage DNAs, pHspAI1 plasmid carrying methylated sequences 5’-G(5mC)GC-3’/3’-CG(5mC)G-5’  and pFsp4HI2 plasmid carrying sites 5’-G(5mC)NGC-3’/3’-CGN(5mC)G-5’ sites .
Figure 2 presents the results of above mentioned DNAs hydrolysis with PkrI.
Figure 2. PkrI hydrolysis of methylated and unmethylated DNA. Electrophoresis in 1% agarose gel. Lanes:
1 - λ phage DNA; 2 - λ DNA + PkrI;
3 - T7 phage DNA; 4 - T7 DNA + PkrI;
5 - pHspAI/GsaI; 6 - pHspAI/GsaI + PkrI;
7 - 1 kb DNA ladder; 8 - pFsp4HI2/DriI;
9 - pFsp4HI2/DriI + BlsI; 10 - pFsp4HI2/DriI + GluI;
11 - pFsp4HI2/DriI + PkrI.
According to Figure 2 PkrI doesn’t cut unmodified λ (line 2) and T7 (line 4) DNA as well as pHspAI DNA (line 6) carrying methylated 5'-GCGC-3' sequences. But PkrI cleaves a linearized plasmid pFsp4HI2 producing DNA fragment ~ 450-500 b.p. in length (lane 11).
The primary structure analysis of pFsp4HI2 has shown a presence of two 5'-GCNGCNGC-3' sites situated from each other at the distance 485 b.p. DNA sequence 5'-GCNGCNGC-3' represents two overlapping sites 5'-GCNGC-3' which are methylated in plasmid pFsp4HI2 with formation DNA sequence 5'-G(5mC)NG(5mC)NGC-3'/3'-CGN(5mC)GN(5mC)G-5' site. Thus, two sites 5'-G(5mC)NG(5mC)-3'/3'-CGN(5mC)G-5' with three 5-methylcytosines are present in pFsp4HI2.
DNA fragment 485 b.p corresponds to the electrophoresis band 450-500 b.p. (Fig.2, lane 11) and we may suggest that PkrI recognizes and cleaves the methylated DNA sequence 5'-GCNGC-3' with three 5-methylcytosines. At the same time Fig.2 shows that BlsI cleaves all sites 5'-G(5mC)NGC-3'/3'-CGN(5mC)G-5' (lane 9) whereas GluI endonuclease doesn’t cut 5'-GCNGC-3' sequences both with two and three 5-methylcytosines (lane 10).
Figure 3 shows the results of hydrolysis of pUC19 and pUC19 methylated with M.CviPI. DNA methyltransferase CviPI methylates the cytosine in 5’-GC-3’ sequence . Plasmids pUC19 has been treated with restriction endonuclease Fsp4HI (lane 2) and methylated pUC19 has been digested with PkrI endonuclease (lane 3).
Figure 3. PkrI cleavage of pUC19 preliminary methylated with M.CviPI. Electrophoresis in 15% PAAG. Lanes:
1 - pUC19/MspI DNA ladder (SibEnzyme);
2 - pUC19 + Fsp4HI;
3 - pUC19/M.CviPI + PkrI.
Figure 3 shows an identity of DNA digestion patterns in case of pUC19 hydrolysis with Fsp4HI (recognition site 5'-GCNGC-3') and in case of pUC19, preliminary methylated at 5'-GC-3' dinucleotides, with PkrI. So, PkrI cuts completely methylated 5’-GCNGC-3’/3’-CGNCG-5’ site with four 5-methylcytosine.
Based on data of Figures 2 and 3 we may suppose that MD endonuclease PkrI recognizes and cleaves methylated 5'-GCNGC-3'/3'-CGNCG-5' sequence carrying at least three 5-methylcytosines.
Determination of PkrI cleavage position. Digestion of synthetic oligonucleotide duplexes has been performed to confirm PkrI recognition sequence and to determine DNA cleavage position. The position of DNA hydrolysis by MD endonuclease PkrI has been determined by comparison of DNA fragments lengths after cleavage of [32P]-labeled synthetic oligonucleotide duplex D1/D2 with PkrI and BlsI endonucleases (see “Materials and Methods”). A partial cleavage of this duplex with ExoIII has been used to prepare DNA marker. Figure 4 shows the autoradiograph of D1/D2 cleavage products electrophoresis. As shown in Fig. 4, the lengths of DNA fragments produced by BlsI and PkrI are identical. Thus, the PkrI and BlsI cleavage position are coincided.
1 – duplex D1*/D2;
2 - duplex D1*/D2, endonuclease PkrI;
3 - duplex D1*/D2, exonuclease III;
4 - duplex D1*/D2, endonuclease BlsI;
Labeled oligonucleotides are shown with the sign *.
Hence, PkrI cleaves the methylated DNA sequence 5'-GCNGC-3' after central nucleotide.
To study PkrI substrate specificity in detail we have performed a hydrolysis of oligonucleotide duplexes with methylated 5'-GCNGC-3'/3'-CGNCG-5' site, carrying different combinations of 5-methylcytosines.
The experimental results are presented in the Figure 5.
1. NN01*/NN02; 2. NN01*/NN02 + PkrI; 3. NN1*/NN2;
4. NN1*/NN2 + PkrI; 5. NN11*/NN22; 6. NN11*/NN22 + PkrI;
7. NN1*/NN22; 8. NN1*/NN22 + PkrI; 9. DD1*/NN02;
10. DD1*/NN02 +PkrI; 11. DD1*/DD2; 12. DD1*/DD2 + PkrI;
13. DD2*/DD1; 14. DD2*/DD1 + PkrI; 15. DD1*/NN1;
16. DD1*/NN1 + PkrI; 17. NN1*/DD1; 18. NN1*/DD1 + PkrI;
19. DD1*/NN22; 20. DD1*/NN22 + PkrI; 21. NN22*/DD1;
22. NN22*/DD1 + PkrI.
According to the data of Figure 5 PkrI doesn’t cleave unmethylated sequence 5'-GCNGC-3'/3'-CGNCG-5' (lane 2) and this sequence with two 5-methylcytosines including 5’-G(5mC)NGC-3’/3’-CGN(5mC)G-5’ (lane 4), 5’-GCNG(5mC) -3’/3’-(5mC)GNGC-5’ (lane 6), 5’-G(5mC)NGC-3’/3’-(5mC)GNCG-5’ (lane 8) and 5’-G(5mC)NG(5mC)-3’/3’-CGNCG-5’ (lane 10). So, PkrI doesn’t cut DNA sequence 5'-GCNGC-3'/3'-CGNCG-5' with any combinations of two 5-methylcytosines.
However, PkrI cleaves 5'-GCNGC-3'/3'-CGNCG-5' sequences with three 5-methylcytosines 5’-G(5mC)NG(5mC)-3’/3’-CGN(5mC)G-5’ (lanes 16, 18) and 5’-G(5mC)NG(5mC)-3’/3’-(5mC)GNCG-5’ (lanes 20, 22). PkrI cuts fully methylated site 5'-GCNGC-3'/3'-CGNCG-5' with four methyl groups as well (lanes 12, 14).
Thus, MD endonuclease PkrI cleaves methylated recognition sequence 5'-GCN↑GC-3'/3'-CG↓NCG-5' carrying at least three 5- methylcytosines as indicated by arrow.
Comparison of known DNA endonucleases properties has shown that Fsp4HI, BisI, BlsI, PkrI and GluI recognize the same DNA sequence 5'-GCNGC-3'/3'-CGNCG-5’ but differ in the cleavage position and a minimal number of 5-methylcytosines in the recognition site. Fsp4HI cleaves only unmethylated sequence 5’-GCNGC-3’ before N, BisI cuts this sequence with at least one 5-methylcytosione before N as well , BlsI digests this site with at least two 5-methylcytosines after N . PkrI cleaves DNA sequence with at least three 5- methylcytosines after N and GluI cleaves a fully methylated sequence before N .
A biological role of such variety of MD endonucleases cleaving methylated sequence 5’-GCNGC-3’ remains unclear.
PkrI may be used in epigenetic studies for analysis of DNA methylation status, in particular, for analysis of human and mammalian DNA.
This work was financially supported by the Ministry of Education and Science of the Russian Federation according of State contract in the context of the Federal Targeted Program "Research and development on priority directions of development for scientific-technological complex of Russia for 2007-2013 years".