FIG. 6.

Drosophila Orc6 protein is important for ORC-dependent DNA binding and DNA replication in vitro. (A) Silver-stained gel of recombinant purified wt and mutant Drosophila ORC proteins. In each lane 100 ng of protein was loaded. (B) DNA binding of ORC6-wt and ORC containing mutant Orc6 proteins. ORC amounts added (50 ng, 100 ng, and 150 ng) are shown above the lanes. (C) DNA binding of the ORC with the Orc6-K76A mutant [ORC(6-K76A)] and the Orc6-S72A mutant [ORC(6-S72A)]) was tested at higher concentrations (150 ng, 300 ng, and 450 ng) of the protein. (D) DNA replication in Drosophila extracts. Xenopus sperm DNA was incubated for 1 h in Drosophila extract (with membranes) at a concentration of 2 to 5 ng/μl in the presence of [³²P]dCTP. Where indicated, extracts were depleted of ORC by using antibodies raised against Orc2 and Orc6. An add-back experiment was performed by the addition of 50, 100, or 150 ng of recombinant ORC proteins to depleted extracts. RE, nondepleted replication extract control (lane 1).