Modulation of Mcm2–7 activity by Cdt1

Abstract

Genome duplication occurs once and only once in each cell cycle. During G1 phase, pre‐replicative complexes (pre‐RC), composed of ORC, Cdc6, Cdt1 and Mcm2–7, are assembled at replication origins. Mcm2–7 is the replicative helicase in eukaryotic cells; however, it does not unwind DNA in the pre‐RC. It is only in S‐phase, after activation of Mcm2–7, that DNA unwinding is detected. Cdt1 co‐purifies from yeast cells in a complex with Mcm2–7. Beyond being an escort for entry of Mcm2–7 into the nucleus and into the pre‐RC, the role of Cdt1 in DNA replication is not known. Using purified components from bacterial expression systems, we assembled Mcm2–7•Cdt1 complexes and compared its activity to that of Mcm2–7 alone. We show that Mcm2–7•Cdt1 has lower ATPase activity than Mcm2–7. In addition, DNA unwinding by Mcm2–7•Cdt1 is significantly lower than by Mcm2–7 alone. Furthermore and consistent with published reports, in vitro pre‐RC reconstitution experiments showed loading of Mcm2–7 to origins was dependent on ORC, Cdc6 and Cdt1. Together, the effects of Cdt1 on Mcm2–7 suggest mechanisms by which Cdt1 may carry out its essential role in the initiation of DNA replication.Research funded by CIHR