FIG. 4.

GST-3a methylates circular plasmid DNA as well as linear DNA fragments in vitro using a restriction enzyme digestion assay. (A) Purified GST fusion protein of Dnmt3a wild type (GST-3a) and mutant (GST-3aMut). Six units of the DNMT1 (New England Biolabs) and approximately 200 ng each of GST-3a and GST3aMut were loaded on an SDS-7% polyacrylamide gel and stained with Coomassie blue stain. (B) Plasmid p220.0 was incubated (+) with GST-3a or GST3aMut, digested with a 10-fold excess of HinP1I, and labeled with [³²P]dCTP using Klenow enzyme as described in Materials and Methods. The complete digestion pattern is shown in the lane that has no protein treatment. The additional bands in the lanes with DNA treated with GST-3a are the HinP1I-resistant bands, indicating DNA methylation. These additional bands are marked by arrowheads. (C) A 467-bp DNA fragment containing three HhaI sites was used as a substrate for GST-3a and GST-3aMut, as described above. The DNA was digested with HhaI before being fractionated on a 1% agarose gel, Southern transferred, and probed with the entire DNA fragment. DNA unmethylated at the HhaI sites gives rise to the 304-bp band after HhaI digestion. The 467-bp band indicates methylation at the three HhaI sites.