FIGURE 9.

The NLS and ORC binding domain in Orc1 are physically and functionally distinct. A, a series of five deletions were constructed that spanned the human Orc1 coding sequence. Two of them are diagrammed along with the bromo-adjacent homology (BAH) required for Orc1 association with chromatin (8), the NLS (40), the AAA+ motif required for ORC(1–5) binding to DNA (11, 13), and the winged-helix motif. The ΔAAA+ deletion mutant was missing residues 482–744, and the ΔW-H deletion mutant was missing residues 783–861. The −NLS mutation replaced ²⁶¹RIKRK²⁶⁵ to ²⁶¹GLEGV²⁶⁵. B, FH-Orc1 was immunoprecipitated (IP) from each of the indicated cell lines with anti-FLAG antibodies; the precipitate was fractionated by SDS-PAGE and then subjected to Western immunoblotting using anti-FLAG, anti-Orc3 and anti-Orc2 antibodies. C, HeLa-RR cells constitutively expressing the indicated FH-Orc1 protein were stained with anti-FLAG antibodies to detect FH-Orc1 and Hoechst to stain DNA.