Structural insights into the Cdt1-mediated MCM2–7 chromatin loading

Abstract

Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2–7 (MCM2–7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410–440)/MCM6(708–821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2–7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2–7, our results suggest that the MCM2–7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2–7 chromatin loading.

Dynamic Association of ORCA with Prereplicative Complex Components Regulates DNA Replication Initiation

“…The recent solution structure of the C-terminal domain of Cdt1 (aa 450 to 557 and aa 420 to 557 of mouse Cdt1) by X-ray crystallography and nuclear magnetic resonance (NMR) revealed the presence of a winged-helix domain that could possibly interact with MCM (23,24). Similarly, NMR studies have revealed the interaction between human Cdt1 (aa 410 to 440) and MCM6 (aa 708 to 821) (29). It is possible that the binding of ORCA to Cdt1 facilitates the loading of MCM2-7, similar to the chromatin remodeler SNF2H, which has been proposed to promote MCM loading via its interaction with Cdt1 (49).…”

From structure to mechanism—understanding initiation of DNA replication

“…Therefore, Cdt1 embraces half of the hexamer (Mcm2, Mcm4, and Mcm6). Interestingly, most of these Cdt1-MCM2-7 interactions have been also observed in the context of the Cdt1-MCM2-7 complex, with the exception of the highly conserved Cdt1-Mcm6 CTD interaction (Wei et al 2010;Liu et al 2012;Fernandez-Cid et al 2013;Yuan et al 2017;Zhai et al 2017), suggesting that this interaction has an important role in OCCM formation and could contribute toward the structural changes in the Mcm NTDs (Fig. 4D).…”

“…Indeed, Cdt1, which is essential for chromatin binding of MCM2-7 (Maiorano et al 2000;Nishitani et al 2000), interacts with the Mcm6 C terminus (Jee et al 2010;Wei et al 2010). Nuclear magnetic resonance (NMR) analysis of the human and budding yeast proteins identified the structure of this important interaction surface, highlighting the Mcm6 WHD for Cdt1 binding (Wei et al 2010;Liu et al 2012). Mutation of conserved amino acids in this domain affects the cell in two ways, blocking MCM2-7 nuclear import and DNA synthesis (Liu et al 2012).…”

“…Nuclear magnetic resonance (NMR) analysis of the human and budding yeast proteins identified the structure of this important interaction surface, highlighting the Mcm6 WHD for Cdt1 binding (Wei et al 2010;Liu et al 2012). Mutation of conserved amino acids in this domain affects the cell in two ways, blocking MCM2-7 nuclear import and DNA synthesis (Liu et al 2012). In vitro analysis revealed that the Mcm6 WHD adopts an autoinhibited conformation that blocks the binding of MCM2-7 to ORC/Cdc6 .…”

A spontaneous Cdt1 mutation in 129 mouse strains reveals a regulatory domain restraining replication licensing

“…Cdc6 and Cdt1 are two essential cofactors for pre-RC establishment, a process known as licensing 4,5 . Differently from other pre-RC components, Cdt1 does not display enzymatic activity and is believed to bridge together the ORC and MCM2/7 complexes during licensing 6,7 .…”