Figure 3.

Licensing and replication activities of various mutant Cdt1 proteins in the presence of Geminin. (A) Western blot analysis of Mcm2, Orc2, Cdc6, Cdt1, and Geminin on chromatin in the egg extract that was either mock-depleted or Cdt1/Geminin double-depleted in the presence of 6 nM wild-type or mutant Cdt1 (R-S, R-C, and RR-AA) proteins with (+) or without (−) Geminin (400 nM). (B) Western blot analysis of indicated proteins in the Cdt1-depleted extract supplemented with 10 nM wild-type or RR-AA Cdt1 protein and Geminin protein at the indicated concentrations. (C) Western blot analysis of indicated proteins in the Cdt1-depleted extract in the presence of various amounts of Geminin (lanes 4–10) or Cdt1/Geminin double-depleted extract supplemented with 10 and 3.3 nM wild-type or RR-AA proteins (lanes 13–25; 3 independent experiments). The licensing activity of the mutant Cdt1/RR-AA is not significantly inhibited by addition of Geminin at a very high concentration. (D) The ability of the wild-type and mutant Cdt1 to restore DNA replication that has been inhibited by an excess Geminin protein was examined. Xenopus egg extract was supplemented with 160 nM Geminin, which resulted in complete inhibition of DNA replication. Then wild-type or mutant Cdt1 proteins were added at the indicated concentrations and incubated in 60 and 90 min, respectively. The replication efficiency is shown by setting the value of [α-³²P]dATP incorporation in the absence of mCdt1 and Geminin at 90 min as 100.