FIGURE 7.

TopoIIα regulates the maintenance of methylation. a, genomic DNAs from wild-type ES cells, Uhrf1 knock-out (KO) ES cells, and Uhrf1 knock-out ES cells reconstituted with SFB-tagged UHRF1 as indicated were digested with HpaII and separated by agarose gel electrophoresis. Southern blotting was performed, and the membrane was blotted using a minor satellite probe. b, the Click reaction was used to detect EdU in HeLa cells treated as indicated. c, Dot blotting was performed using genomic DNA extracted from HeLa cells treated as indicated. The antibody against 5-methylated cytosine was used. d, nascent DNA was purified from HeLa cells treated as indicated. PCR was used to amplify three genomic locations as shown. e and f, bisulfite conversion was performed using the purified nascent DNA from HeLa cells treated with ICRF-193 or ICRF-187 as indicated, and PCR was used to amplify genomic loci for COBRA (e) and bisulfite sequencing analysis (f). In e, bands corresponding to methylated (M) and unmethylated (U) DNA are labeled. The asterisk indicates nonspecific PCR bands. In f, methylated and unmethylated cytosines are represented by black and white circles, respectively.