FIG. 4.

Spb70 genetically and physically interacts with ORC proteins. (A) Genetic interaction. Wild type (wt), orp1-4, spb70-U03, and double mutant spb70-U03 orp1-4 were cultured in liquid YES medium at 25°C. Equal numbers of log-phase cells were spotted on YES plates in threefold serial dilution and incubated at 25 and 30°C for 4 days. (B) Spb70 protein physically associates with Orp1/Orc1. HA-orp1^+/orc1 and two-myc-six-His-spb70⁺ HA-orp1⁺ strains were grown at 25°C in EMM to mid-log phase and whole-cell extracts (W.C.E.) were prepared from each strain. Presence of Spb70 and Orp1 in whole-cell extracts was detected by anti-myc and anti-HA antibody, respectively (W.C.E. panel). Ni bead pull-down fractions were Western blotted with anti-myc for Spb70 and anti-HA for Orp1 (Ni-beads panel). (C) Spb70 protein physically associates with Orp2/Orc2, and a mutation in spb70⁺ compromises the interaction. Wild-type cells containing two-myc-six-His-spb70⁺ and orp2⁺-three-FLAG and the mutant containing two-myc-six-His-spb70-U03 and orp2⁺-three-FLAG were cultured at 36°C. Ni bead pull-down fractions from each strain's chromatin fraction were Western blotted with anti-myc and anti-FLAG for Spb70 and Orp2, respectively. The Orp2 proteins in the chromatin fraction of wild-type and mutant cells used for the Ni bead pull-down experiment are shown in the bottom panel.