INTRODUCTION

Endonucleases of restriction are widely used currently in molecular biology. Restriction–modification systems are used by many prokaryotes to protect their own genome from foreign genetic material entering the cell [1, 2]. As a rule, the restriction–modification system includes two enzymes: DNA methyltransferase, which methylates certain recognition sites of the host DNA, thereby protecting it from the action of its own restrictases, and restriction endonuclease that cleaves unmethylated foreign DNA. In particular, type II restriction endonucleases differ from others in that they cleave DNA within the recognition site or in close proximity to it; they function as homodimers and do not require ATP hydrolysis for activity. They recognize palindromic sequences of 4–8 nucleotides and cleave them with the formation of a double-stranded gap.

The endonucleases capable to selectively recognize and cleave DNA containing different methylated bases are of particular interest for epigenetic studies. Some of them, for example, endonuclease MspJI, that cleaves DNA containing 5-methylcytosine (mC), are typical restriction endonucleases with a characteristic catalytic domain [3]. Others, such as endonuclease DpnI, specific for DNA containing N6-methyladenine, have a unique structure and, in all likelihood, perform the cell functions that are not associated with protection against foreign genetic material [4]. The recently discovered enzyme GlaI, isolated from the bacterial isolate GL29 of an unidentified species of the Microbacteriaceae family of the order Actinomycetales, belongs to such atypical endonucleases [5, 6]. GlaI endonuclease is capable of cleaving the 5'‑GmC↓GC-3' recognition site containing mC, but is not capable of hydrolyzing unmodified DNA; it is now increasingly being used to analyze the methylation status of eukaryotic DNA [6–13]. Although the sequence 5'-GmCGC-3' is the minimal recognition site of the endonuclease GlaI, the most efficient cleavage of DNA occurs at sites containing at least three bases mC or hmC; and the palindromic sequence 5′‑GmCGmC-3′/3′-mCGmCG-5′ with four bases mC shows the maximal level of cleavage [6].

DNA methylation in CpG dinucleotides to form mC (Fig. 1a) is a special way of controlling the activity of a wide range of genes, which has maximally developed in higher eukaryotes [14, 15]. The effect of mC on gene activity is mainly mediated by proteins containing a methyl binding domain, a portion of the polypeptide chain capable of recognizing a fully methylated CpG dinucleotide in DNA [16]. The proteins containing it (MeCP2, MBD1, MBD2, MBD3, etc.) form complexes with histone deacetylases or possess the activity of histone-specific methyltransferase, chromatin remodeling factors, etc. [17]. These functional interactions lead to condensation of chromatin in the region of methylated CpG-rich segments and suppression of transcription. The level of methylation of promoter regions shows a strong negative correlation with transcriptional activity [18, 19]. Recent studies have shown that the oxidized 5-methylcytosine derivative 5-hydroxymethylcytosine (hmC; Fig. 1a), formed due to the directed oxidation of mC in CpG sites by TET1, TET2 and TET3 dioxygenases, also plays an epigenetic role in the mammalian genome [20]. The promoter regions of actively expressed genes are enriched not with mC but with hmC, which is considered as an activating epigenetic marker [21, 22]. Proteins that specifically bind hmC in DNA were found [23, 24], but there is no clear understanding of how this base affects transcriptional activity yet.

Fig 1.
figure 1

Cleavage of substrates containing various combinations of mC and hmC in the recognition site of endonuclease GlaI. (a) The structures of cytosine, 5-methylcytosine and 5-hydroxymethylcytosine. dR is the residue of deoxyribose in DNA. (b) The general sequence of the substrates used in the study. A fragment of the sequence in which the bases C were replaced by mC and hmC in various combinations is highlighted. The radioactive label in all cases was in the upper specified chain. (c), the electropherogram of a representative experiment. The enzyme was present in the tracks with even numbers. The arrows indicate the noncleaved substrate (S) and the cleavage product (P). In the shown sequences, M is for mC and H is for hmC.

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The discovery of the existence of hmC and its special role in the epigenome of higher eukaryotes posed a question about the specificity of molecular biological tools used for the analysis of epigenetic methylation. In particular, it is not clear with what efficiency mC-specific restriction endonucleases are capable to cleave the DNA containing hmC recognition sites or various combinations of mC and hmC. In this study, we compared the efficiency of the cleavage of methylated and hydroxymethylated DNA with methyl-dependent DNA endonuclease GlaI.

RESULTS AND DISCUSSION

Methyl-dependent DNA endonuclease GlaI has recently become popular as an epigenome analysis tool, suitable for the study of cytosine methylation in the DNA of both mammals and plants [6–13]. Although this enzyme is one of the few endonucleases available, which requires mC in the 5'-GCGC-3' recognition site, its activity varies quite strongly depending on the total amount of mC in this site. According to the literature, the difference between activities of the worst and best substrates, 5'-GmCGC-3'/3'-CGmCG-5' and 5'-GmCGmC-3'/3'-mCGmCG-5', is about 20 times [6]. GlaI recognizes with less efficiency the sites other than 5'-GCGC-3' of the general form 5'‑RCGY-3' (R is a purine base and Y is a pyrimidine base) [6].

The ability of the GlaI enzyme to cleave substrates containing some combinations of C, mC and hmC in an optimal recognition site was investigated (Fig. 1b). The observed efficiency to cleave substrate 5′-GmCGmC-3′/3′-mCGmCG-5′ (Fig. 1, lanes 7–8 and 15–16) compared to other substrates confirmed that the recognition site containing four mC is optimal for hydrolysis by endonuclease GlaI. Replacement of all mC by hmC reduced the amount of the cleavage product by two times (Fig. 1, lanes 13–14). Replacement of one mC of the complementary chain to C or hmC in the immediate vicinity of the cleavage site significantly reduced the cleavage efficiency (Fig. 1, lanes 5–6 and 21–22). Further deterioration of cleavage was observed after the replacement of two mC to hmC directly around the hydrolyzed bond (Fig. 1, lanes 25–26). On the other hand, the replacement of one edge mC in the recognition site of a complementary chain with an unmethylated nucleotide or its oxidized derivative also affected the cleavage efficiency, but to a lesser extent (Fig. 1, lanes 17–20). An unmodified recognition site, or a site containing no more than 2 modified nucleotides, was hardly cleaved by the enzyme (Fig. 1, lanes 1–4 and 9–10).

The study was conducted to determine the stationary kinetics of cleavage of DNA substrates containing the sites 5′-GmCGmC-3′/3′-mCGmCG-5′ and 5′‑GhmCGhmC-3′/3′-hmCGhmCG-5′ and the kinetic parameters KM and Vmax of the reaction for a quantitative comparison of efficiency of recognition of mC and hmC (Table 1). It can be seen that the maximal rate for a fully methylated substrate after normalization of Vmax by the amount of the enzyme was approximately twice as high as for a fully hydroxymethylated substrate. The Michaelis constant for a fully hydroxymethylated substrate was approximately four times higher than for a fully methylated substrate, indicating less stability of the enzyme-substrate complex in the presence of hmC bases in the site of recognition. The enzyme specificity proportional to the Vmax/KM ratio was approximately 8.4 times higher for a fully methylated recognition site compared to a fully hydroxymethylated site.

Table 1.   Kinetic constants of the GlaI endonuclease cleavage reaction at the fully methylated and fully hydroxymethylated 5'-GCGC-3' site
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Thus, it can be concluded from the comparison of the cleavage efficiency of various combinations of C, mC and hmC that the hmC bases are recognized by the GlaI enzyme somewhat worse than the mC bases. This may be due to the presence of an additional hydroxyl group of hmC in the large DNA groove. According to X-ray diffraction analysis, it does not interact with other atoms in the groove [25] and therefore can easily come into contact with donors and acceptors of hydrogen bonds in the protein; in addition, its volume can sterically affect the binding of GlaI to DNA. The central position of the sequence 5′-GCGC-3′ is of the utmost importance for recognition by the enzyme GlaI, while modification of the edge cytosines contributes to the recognition less. It is, in all probability, due to the degeneracy of the recognition site of the enzyme GlaI, which cleaves with some effectiveness all the 5′-RCGY-3′ sequences containing mC [6]. The ability of GlaI to recognize hmC in sequences other than optimal 5′-GCGC-3′ may be the subject of further research. In general, it can be noted that the less effective cleavage of hmC-rich DNA sites by GlaI should be taken into account in any analysis of epigenetic status with this enzyme.

EXPERIMENTAL

Materials. Oligodeoxyribonucleotides (Fig. 1b) were synthesized by phosphoramidite solid-phase method on an ASM-800 automatic synthesizer (OOO Biosset, Russia) using commercially available phosphoramidites of mC and hmC (Glen Research, United States). Polynucleotide kinase of bacteriophage T4 was from Biosan (Russia) and DNA endonuclease GlaI was from Sibenzyme (Russia).

DNA substrates of endonuclease GlaI were obtained by introduction of the 32P-label at the 5′-end of oligonucleotides by standard methods using γ[32P]-ATP and polynucleotide kinase of bacteriophage T4 [26]. Labeled oligonucleotides were separated from the remaining γ[32P]-ATP by reversed phase chromatography on the C18 NenSorb sorbent (DuPont, United States) and annealed with a double molar excess of the necessary unlabeled complementary chain.

To study the activity of GlaI endonuclease, the reaction mixture contained 33 mM Tris acetate (pH 7.9), 10 mM Mg(CH3COO)2, 66 mM CH3COOK, and 1 mM dithiothreitol (Sibenzyme buffer Y), the substrate at a concentration of 50 nM or 100 nM and 0.35 U/µL of the enzyme. The mixture was incubated at 30°C for 30 min; the reaction was stopped by adding an equal volume of 80% formamide containing 20 mM sodium ethylenediaminetetraacetate, 0.1% xylene cyanol (w/v), and 0.1% bromophenol blue (w/v), and heated at 95°C for 5 min. The reaction products were analyzed by electrophoresis in 20% polyacrylamide gel containing 8 M urea, followed by radioluminescent scanning with Typhoon FLA 9500 (GE Healthcare, United States). The results were calculated with the Quantity One v 4.6.3 program (Bio-Rad, United States).

To determine the parameters of stationary kinetics of DNA-endonuclease GlaI with substrates 5′-GmCGmC-3′/3′-mCGmCG-5′ and 5′-GhmCGhmC-3′/3′-hmCGhmCG-5′, the reaction mixture contained 3 mM Tris-acetate (pH 7.9), 10 mM Mg(CH3COO)2, 66 mM CH3COOK, 1mM dithiothreitol, the substrate at a concentration of 10–700 nM and the enzyme at a concentration of 0.02 or 0.05 U/µL for methylated and hydroxymethylated substrate, respectively. The mixture was incubated at 30°C for 5 min. The reaction was stopped; the products were separated and analyzed as described above.