Fig 1: Biochemical methylation of CpG dinucleotide‐containing nucleosomal DNA (CpG146). Lanes are as follows: M, 10‐bp DNA ladder (Thermo Fisher Scientific, Waltham, MA, USA; cat. 10821‐015); (+), presence; and (−), absence of the respective enzyme shown on the left. (A) Comparison of the CpG methyltransferase M.SssI enzymatic activities. CpG146 DNA was methylated with M.SssI enzymes as follows: lanes 3–5, M.SssI purchased from New England Biolabs (NEB, cat. M0226M); and lanes 6–11, the M.SssI protein purified in this study. In lanes 1 and 2, CpG146 DNAs were incubated in the methylation reaction buffer, in the absence of M.SssI. Each DNA sample was methylated with an increasing amount of M.SssI, and the DNA from each reaction was purified, digested with the methyl‐sensitive restriction endonuclease Eco72I, and electrophoresed. The specific units of the NEB M.SssI enzyme used in each reaction are as follows: lane 3, 2.0 units·μg−1 DNA; lane 4, 3.0 units·μg−1 DNA; and lane 5, 4.0 units·μg−1 DNA. The amount of the purified M.SssI protein used in each reaction is as follows: lane 6, 0.032 μg·μg−1 DNA; lane 7, 0.063 μg·μg−1 DNA; lane 8, 0.13 μg·μg−1 DNA; lane 9, 0.25 μg·μg−1 DNA; lane 10, 0.50 μg·μg−1 DNA; and lane 11, 1.0 μg·μg−1 DNA. The positions of the 146‐bp CpG146 nucleosomal DNA, the half unit of CpG146, and the digested DNAs are shown by a red arrow, a black arrow, and a gray square bracket, respectively. (B) Confirmation of the CpG‐methylated CpG146 DNA by Eco72I digestion. The digestion was performed with 20 units·μg−1 DNA (1.8 units·pmol−1 DNA). Lanes 1 and 3, unmodified CpG146 DNA; lanes 2 and 4, M.SssI‐treated CpG146 DNA. In lanes 3 and 4, the nucleosomal DNA was subsequently incubated with the methylation‐sensitive restriction enzyme Eco72I. (C) Confirmation of the CpG‐methylated CpG146 DNA by MspJI digestion. The digestion was performed with 5 units·μg−1 DNA (0.45 units·pmol−1 DNA). Lanes 1 and 3, unmodified CpG146 DNA; lanes 2 and 4, M.SssI‐treated CpG146 DNA. In lanes 3 and 4, the nucleosomal DNA was subsequently incubated with the methylation‐dependent restriction enzyme MspJI.
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Unit Definition:One unit is defined as the amount of enzyme required to protect 1 µg of lambda DNA in a total reaction volume of 20 µl in 1 hour at 37°C against cleavage by BstUI restriction endonuclease.