Characterization of the 5-hydroxymethylcytosine-specific DNA restriction endonucleases

Abstract

In T4 bacteriophage, 5-hydroxymethylcytosine (5hmC) is incorporated into DNA during replication. In response, bacteria may have developed modification-dependent type IV restriction enzymes to defend the cell from T4-like infection. PvuRts1I was the first identified restriction enzyme to exhibit specificity toward hmC over 5-methylcytosine (5mC) and cytosine. By using PvuRts1I as the original member, we identified and characterized a number of homologous proteins. Most enzymes exhibited similar cutting properties to PvuRts1I, creating a double-stranded cleavage on the 3' side of the modified cytosine. In addition, for efficient cutting, the enzymes require two cytosines 21-22-nt apart and on opposite strands where one cytosine must be modified. Interestingly, the specificity determination unveiled a new layer of complexity where the enzymes not only have specificity for 5-β-glucosylated hmC (5βghmC) but also 5-α-glucosylated hmC (5αghmC). In some cases, the enzymes are inhibited by 5βghmC, whereas in others they are inhibited by 5αghmC. These observations indicate that the position of the sugar ring relative to the base is a determining factor in the substrate specificity of the PvuRts1I homologues. Lastly, we envision that the unique properties of select PvuRts1I homologues will permit their use as an additive or alternative tool to map the hydroxymethylome.

Figure 1.

Purified homologues and gel filtration analysis of the homologues. (A) The following homologues were run on a Tris–glycine 4–20% gel as an example of the relative purity of the proteins. Lane M is the ColorPlus protein ladder (NEB # P7710). Lane 1: AbaTI, lane 2: AcaPI, lane 3: AbaUI, lane 4: AbaDI and lane 5: YrkI. (B) Analytical size exclusion chromatography of the homologues. The two asterisks along the standard curve indicate the narrow range of 1.62–1.69 in which all the homologues eluted. The molecular weights were determined relative to their elution volume against that of the molecular weight standards and are summarized in Table 3.

Figure 2.

Relative selectivity of PvuRts1I homologues. Selectivity was determined on DNA with different modified cytosines: dcm⁻ (unmodified cytosine), XP12 (methylated cytosines), T4wt (hydroxymethylated cytosines), T4α (α-glucosylated hydroxymethylated cytosines), T4β (β-glucosylated hydroxymethylated cytosines).

Figure 3.

Cleavage site determination of the PvuRts1I homologues. (A) The left side of the figure shows the sequence of the hemi-glucosylhydroxymethylated 3′FAM-labeled 54-bp oligonucleotide used to determine the cleavage site on the same strand of the modification. The position of the modified cytosine along with the expected cut sites on the top and bottom strand is indicated. For the right side of the figure, fragments from AbaAI, AbaUI and BbiDI digestion, along with oligonucleotide markers of 15 and 14 nt were resolved on a denaturing polyacrylamide gel. Recognition of the modification by the homologues resulted in two labeled fragments of 37(+/−) nt, from cleavage on the opposite strand of the modification, and 15(+/−) nt, from cleavage on the same strand of the modification. (B) The left side of the figure shows the sequence of the fully glucosylhydroxymethylated 5′FAM-labeled oligonucleotide used to determine the cleavage site on the opposite strand of the modification. Three expected cut site scenarios are shown: (1a, 1b) recognition of the modification would result in a cleavage to the 3′ side of the modification, yielding two labeled fragments of 39(+/−) nt, from cleavage on the same strand of the modification, and 17(+/−) nt, from cleavage on the opposite strand of the modification; (2) recognition of both modifications will result in a right-hand cleavage for the top-strand modification and a left-hand cleavage for the bottom-strand modification yielding two labeled bands of 17(+/−) nt, resulting from cleavage on the opposite strands of the modifications. The gel on the right side of Figure 3B shows the fragments from AbaAI, AbaUI and BbiDI digestion, along with an oligonucleotide marker of 18 nt. Recognition of the substrate by the homologues shows a mixture of labeled fragments of 39(+/−) and 17(+/−), resulting from cleavage scenarios 1a, 1b and 2. The 39(+/−) nt fragment is labeled as an intermediate because if the reaction went to completion, only the 17(+/−) nt fragments will be observed. Due to the resolving power of the gel, only the size of the smaller fragments of DNA could be accurately determined. However, by simply subtracting the smaller fragment from the total length, the size of the larger fragment could also be derived. The cut site for all the enzymes is predominantly N_11–13/N_9–10 (Table 4). The digestion range for the cut site is owing to some enzymes exhibiting minimal base wobbling.

Figure 4.

Activity of enzymes on different modified oligonucleotides. The sequence of the oligonucleotides can be found in Table 2. (A) Schematic of the five modified oligonucleotides used for the activity determination; A, C/C, C, 5mC and 5hmC. The two indicated residues on each substrate are 22 nt apart. (B) The extent of double strand cleavage on A, C/C, C, 5mC and 5hmC for each of the homologues is shown. All of the homologues have the highest activity on a substrate containing two 5hmC modifications, 22 nt apart (5hmC, blue). The activity decreases as the modification on the opposite strand changes from 5mC to C and there is no detectable cutting for most of the homologues in the absence of cytosine (A, yellow). The activities are normalized to cutting on 5hmC.