Cryo-EM structures of the eukaryotic replicative helicase bound to a translocation substrate

Abstract

The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.

New insights into the GINS complex explain the controversy between existing structural models

“…These data confirm that in solution the major GINS population corresponds to a tetramer with a shape consistent with the crystal structures111213. The EM structure of the CMG complex also confirms that the GINS crystallographic tetramer can also easily fit into the CMG density without conformational changes45.…”

“…The helicase core of the replicative fork is formed by the CMG complex, which includes the MCM2–7 helicase, Cdc45 and the GINS complex3. The MCM complex is loaded onto origins as an inactive helicase and is activated upon Cdc45 and GINS binding45.…”

Ufd1-Npl4 Recruit Cdc48 for Disassembly of Ubiquitylated CMG Helicase at the End of Chromosome Replication

“…In this context, it is interesting to note that lysine 29 of Mcm7 is located within “sub-domain A,” which in archaeal MCM helicases undergoes a 150° rotation between different structural forms (Chen et al., 2005, Miller et al., 2014). More recently, electron microscopy structures of yeast Mcm2–7 (Li et al., 2015) and Drosophila CMG (Abid Ali et al., 2016) indicate that sub-domain A of Mcm7 rotates significantly between the loaded inactive Mcm2–7 complex and the active CMG helicase. It thus seems plausible that structural changes in sub-domain A of Mcm7 might represent a conformational switch on the CMG helicase during termination, for example, revealing a docking site for the ubiquitin ligase or otherwise controlling the access of the ligase to K29.…”

Architecture of the Saccharomyces cerevisiae Replisome

“…10.1a–c) (Yuan et al 2016). The secondary pore also disappears in a medium resolution cryo-EM map of Drosophila CMG at 7–10 Å resolution (Abid Ali et al 2016). The Mcm2-7 core in the CMG forms a two-tiered ring structure: an N-terminal domain (NTD) tier composed of a helical subdomain, a Zn-binding motif, and an oligonucleotide/oligosaccharide-binding (OB) motif, while the C-terminal domain (CTD) tier contains the AAA+ motors.…”

“…In the medium resolution cryo-EM structures of the Drosophila CMG, density corresponding to six nucleotides of ssDNA was observed either inside the CTD-tier motor ring or inside the NTD-tier ring (Abid Ali et al 2016). This observation is consistent with CMG acting as an ssDNA translocase.…”