Cdt1p, through its interaction with Mcm6p, is required for the formation, nuclear accumulation and chromatin loading of the MCM complex

Abstract

SummaryRegulation of DNA replication initiation is essential for the faithful inheritance of genetic information. Replication initiation is a multi-step process involving many factors including ORC, Cdt1p, Mcm2-7p and other proteins that bind to replication origins to form a pre-replicative complex (pre-RC). As a prerequisite for pre-RC assembly, Cdt1p and the Mcm2-7p heterohexameric complex accumulate in the nucleus in G1 phase in an interdependent manner in budding yeast. However, the nature of this interdependence is not clear, nor is it known whether Cdt1p is required for the assembly of the MCM complex. In this study, we provide the first evidence that Cdt1p, through its interaction with Mcm6p with the C-terminal regions of the two proteins, is crucial for the formation of the MCM complex in both the cytoplasm and nucleoplasm. We demonstrate that disruption of the interaction between Cdt1p and Mcm6p prevents the formation of the MCM complex, excludes Mcm2-7p from the nucleus, and inhibits pre-RC assembly and DNA replication. Our findings suggest a function for Cdt1p in promoting the assembly of the MCM complex and maintaining its integrity by interacting with Mcm6p.

Cdt1 stabilizes an open MCM ring for helicase loading

“…Figure 1a (arrowheads) shows that both Mcm6, as previously shown15, and Mcm2 were co-immunoprecipitated with Cdt1 using an anti-Cdt1 antibody in a Cdt1-dependent manner. We next used glycerol gradients to examine complex formation between Cdt1, Mcm2 and Mcm6 (Supplementary Fig.…”

Multiple Functions for Mcm2–7 ATPase Motifs during Replication Initiation

“…7). Consistent with direct interactions between Mcm6 and Cdt1 (Wu et al, 2012), MCM6RA (and to a lesser extent its ATPase site counterpart, MCM2KA) showed reduced recruitment of Cdt1 and Mcm2-7 during OCCM complex formation (Fig. 2 and S2C).…”

An Essential and Cell-Cycle-Dependent ORC Dimerization Cycle Regulates Eukaryotic Chromosomal DNA Replication

“…Yeast cell cycle block and release with 5 mg/ml a-factor (G 1 phase), 150 mM hydroxyurea (S phase) or 10-20 mg/ml nocodazole (M phase) at permissive/non-permissive temperatures were carried out as described previously (Wu et al, 2012). Flow cytometry was done as previously described (Zhang et al, 2002).…”