Involvement of Human MCM8 in Prereplication Complex Assembly by Recruiting hcdc6 to Chromatin

Abstract

The MCM2-MCM7 complex is an essential component of the prereplication complex (pre-RC), which is recruited by the cdc6 and cdt1 proteins to origins of DNA replication during G₁ phase. Here, we report that the accumulation on chromatin of another member of the MCM protein family, human MCM8 (hMCM8), occurs during early G₁ phase, before the hMCM2-hMCM7 complex binds. hMCM8 interacts in vivo with two components of the pre-RC, namely, hcdc6 and hORC2. Depletion of endogenous hMCM8 protein by RNA interference leads to a delay of entry into S phase, suggesting a role for hMCM8 in G₁ progression. Furthermore, down-regulation of hMCM8 also leads to a reduced loading of hcdc6 and the hMCM2-hMCM7 complex on chromatin. These results suggest that hMCM8 is a crucial component of the pre-RC and that the interaction between hMCM8 and hcdc6 is required for pre-RC assembly.

ACKNOWLEDGMENTS

We are grateful to Fumio Hanaoka for providing antiserum against human MCM10 and Bruce Stillman for supplying Cdc6 and ORC2 expression plasmids and antiserum against human ORC2. We thank the members of our lab for discussion and Irina Kotova for critically reading the manuscript.