Figure 3.

M.HpaII modification at the −5 through +38 locations inhibits ORC DNA binding. A–G, electrophoretic mobility shift assays were used to compare ORC DNA binding to ARS1 DNA templates with or without M.HpaII cross-linking at the indicated positions. Control experiments with wild-type (WT) unmodified ARS1 DNA or a mutant ARS1 DNA lacking ORC-binding sites (A-B2-) are shown in A. For the remaining panels, the site of the 5FdC-modified M.HpaII-binding site and templates that have been cross-linked to M.HpaII are indicated. Note that binding to M.HpaII-modified DNA results in a supershifted complex relative to ORC binding to the same DNA without M.HpaII cross-linking (see C and E). Low levels of shifted DNA in the M.HpaII-cross-linked samples at high levels of ORC in D and F (marked with an asterisk) are due to ORC binding to a small amount of unmodified DNA present in these DNA preparations (note the lack of a supershift relative to the unmodified DNA samples).