Figure 3.
Recombinant HsORC binds DNA with little sequence specificity. Recombinant HsORC was incubated with a fixed quantity of radiolabeled lamin B2 ori II probe and various amounts of different competitor DNAs in the presence of ATP as indicated. The extent of binding of the radioactive lamin B2 ori II DNA was measured by the nitrocellulose filter-binding assay. As in Figure 2, the fold competitor is in terms of concentration of nucleotides. (A) Competition with human lamin B2 origin DNA. Overlapping fragments that constitute the human lamin B2 origin were generated by PCR and used as competitors. (B) Competition with Chinese hamster DHFR origin DNA. Fragments that span DHFR oriβ were synthesized by PCR and used as competitors. (C) Competition with nonorigin DNA. A fragment of pUC19 and three fragments of pBS (I, II, and III) were used as competitors.