Open-ringed structure of the Cdt1–Mcm2–7 complex as a precursor of the MCM double hexamer

Abstract

The minichromosome maintenance complex (MCM) hexameric complex (Mcm2-7) forms the core of the eukaryotic replicative helicase. During G1 phase, two Cdt1-Mcm2-7 heptamers are loaded onto each replication origin by the origin-recognition complex (ORC) and Cdc6 to form an inactive MCM double hexamer (DH), but the detailed loading mechanism remains unclear. Here we examine the structures of the yeast MCM hexamer and Cdt1-MCM heptamer from Saccharomyces cerevisiae. Both complexes form left-handed coil structures with a 10-15-Å gap between Mcm5 and Mcm2, and a central channel that is occluded by the C-terminal domain winged-helix motif of Mcm5. Cdt1 wraps around the N-terminal regions of Mcm2, Mcm6 and Mcm4 to stabilize the whole complex. The intrinsic coiled structures of the precursors provide insights into the DH formation, and suggest a spring-action model for the MCM during the initial origin melting and the subsequent DNA unwinding.

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).…”

“…In the OCCM, the helicase is associated with its DNA loader (ORC/Cdc6), and the dsDNA is already inside the central channel of MCM2-7, although the DNA entry gate between the Mcm2 and Mcm5 interface still remains partially open (Sun et al 2013;Yuan et al 2017). Crucially, while, in the MCM2-7/Cdt1 precursor, the six Mcm proteins are arranged in a spiral structure, in the OCCM complex, they are arranged in a plane similar to those in the MCM2-7 DH and CMG Zhai et al 2017). Importantly, Cdt1, which is essential for ORC/Cdc6 and MCM2-7 association, interacts with both the core helicase and the DNA loader (Chen et al 2007;Fernandez-Cid et al 2013;Sun et al 2013;Ticau et al 2015;Yuan et al 2017).…”

“…Crystallography and cryo-EM have generated near-atomic resolution structures of several large protein complexes, including Drosophila melanogaster and Homo sapiens ORC (Bleichert et al 2015;Tocilj et al 2017); Saccharomyces cerevisiae MCM2-7 and MCM2-7/Cdt1 (Zhai et al 2017), OCCM complex , and MCM2-7 DH ; S. cerevisiae and D. melanogaster CMG (Abid Ali et al 2016;Yuan et al 2016;Georgescu et al 2017); S. cerevisiae Ctf4 trimer (Simon et al 2014), polymerase ε (Hogg et al 2014), and polymerase δ (Swan et al 2009); and H. sapiens Mcm2-H3/H4 (Huang et al 2015;Richet et al 2015). Here we concentrate on four key complexes involved in initiation of DNA replication; namely, S. cerevisiae ORC, OCCM, MCM2-7 DH, and CMG (Fig.…”

“…MCM2-7 is the core of the replicative DNA helicase and consists of six subunits that have a spiral arrangement with a gap at the Mcm2/5 interface Ticau et al 2017;Zhai et al 2017). During helicase loading, ORC, Cdc6, and Cdt1 insert DNA through the Mcm2/Mcm5 gate into the complex, and the MCM2-7 ring closes partially around dsDNA (Sun et al 2013;Samel et al 2014;Zhai et al 2017). Crucially, the MCM2-7 recruitment process depends on an Mcm6-Cdt1 interaction, which alleviates an autoinhibitory activity of the Mcm6 C terminus .…”

“…The OB-fold is interrupted by a DNA-binding N-terminal hairpin (NtHp) and a zinc finger (ZF). One of the main functions of the ZFs is to form the DH interface, which gives rise to the extraordinary salt stability of the complex Zhai et al 2017). N-terminal extensions (NTEs) (Fig.…”