FIG. 3.

myc origin DNA rescues Pre-RC assembly on CpG-methylated DNA. (A) ORC2, MCM3, RCC1, and H2A association with unmethylated and methylated lambda DNA assembled in membrane-free egg extracts. A total of 25 ng of methylated and unmethylated lambda was incubated for 30 min in 10 μl of membrane-free cytosol. Western blot analysis (left) or total protein staining with Coomassie blue (right) is shown. (B) ORC2 association with methylated and unmethylated plasmids containing either no insert, a 1,263-bp cdc45 insert, or a 1,704-bp cdc45 insert. A total of 125 ng of unmethylated and methylated plasmid was assembled in 50 μl of membrane-free egg cytosol for 30 min. DNA recovery was monitored by staining a standard agarose gel with Sybergold reagent. (C) ORC2 association was determined as in panel B except that plasmid containing either no insert or myc origin DNA was used.