Cryo-EM structure of a licensed DNA replication origin

Abstract

Eukaryotic origins of replication are licensed upon loading of the MCM helicase motor onto DNA. ATP hydrolysis by MCM is required for loading and the post-catalytic MCM is an inactive double hexamer that encircles duplex DNA. Origin firing depends on MCM engagement of Cdc45 and GINS to form the CMG holo-helicase. CMG assembly requires several steps including MCM phosphorylation by DDK. To understand origin activation, here we have determined the cryo-EM structures of DNA-bound MCM, either unmodified or phosphorylated, and visualize a phospho-dependent MCM element likely important for Cdc45 recruitment. MCM pore loops touch both the Watson and Crick strands, constraining duplex DNA in a bent configuration. By comparing our new MCM-DNA structure with the structure of CMG-DNA, we suggest how the conformational transition from the loaded, post-catalytic MCM to CMG might promote DNA untwisting and melting at the onset of replication.

Fig. 1

Structure of the unmodified and DDK-phosphorylated MCM double hexamer. a Scheme for the loading and phosphorylation of the MCM double hexamer on biotinylated duplex DNA. DDK was removed by high-salt wash. MCM particles retained on digested DNA were used for cryo-EM experiments. b Silver-stained SDS–PAGE gel of (lane 1) unmodified and (lane 2) phosphorylated MCM compared to the DDK input (lane 3). No trace of DDK is found in the MCM lanes. A black arrow points at the shifted phospho-Mcm6. c 2D class averages of the unmodified (first row) and DDK phosphorylated MCM double hexamer (second row). The third row contains a difference image obtained by subtracting unphosphorylated from phosphorylated MCM. Purple arrowheads point to the absence (hollow) or presence (filled) of additional density surrounding the double-hexamer interface in the unmodified and DDK phosphorylated samples, respectively. d 4.65 Å resolution (unsharpened map) of the DDK phosphorylated MCM double-hexamer with docked in atomic coordinates of yeast MCM (PDB: 3ja8). e A cut-through view of the DDK phosphorylated MCM double hexamer (unsharpened map) reveals a narrowing of the channel at the N-terminal domain interface and a poor occupancy of DNA. f Phosphorylated MCM displayed at σ = 0.37, superposed to a difference map displayed at σ = 0.08 (purple). To obtain the difference map the unmodified MCM was subtracted from the DDK phosphorylated MCM. g Cartoon depicting the effect of MCM phosphorylation by DDK. Phosphorylation increases rigidity to N-terminal Mcm4-6, which become visible upon averaging

Fig. 2

Near atomic resolution model of MCM and nucleotide occupancy of the ATPase motor. a 4.3 Å resolution structure of the phosphorylated MCM and a detail of the dimerization interface shown on the right. b On the left: atomic model of the MCM ATPase motor, overlaid with the nucleotide density recovered in between AAA+ protomers. Mcm5-3 and Mcm5-2 contain strong nucleotide density peaks. The same ATPase site profile can be observed for the unphosphorylated form of MCM. Mcm2-6 is nucleotide free and in a relaxed configuration favouring ADP release. On the right: atomic model of ATP-CMG. Mcm5-3, Mcm2-5 and Mcm6-2 are bound to ATP

Fig. 3

Signal subtraction of ATPase motor allows structural characterization of the DNA-bound double hexamer. a Signal subtraction scheme, based on Bai et al.. The full 3D model is composed of the AAA+ ATPase and N-terminal tiers. A mask was created for the ATPase tier, which was applied to the experimental images (particles) for subtraction. The remaining density corresponds to N-terminal tiers and DNA (if present) that would emanate from the channel. b Signal subtracted particles were subjected to 2D classification. Classes showing helical DNA density protruding from the N-terminal tiers were selected and reverted to their original full density before 3D refinement. c Cryo-EM map of the DNA-bound, unphosphorylated MCM double hexamer

Fig. 4

Structure of the MCM double hexamer bound to duplex DNA. a Cut-through view of the cryo-EM map (6.9 Å resolution) with the refined MCM atomic coordinates. Both Watson and Crick strands are touched by MCM pore loops in each single hexamer. b 62 base pairs of duplex DNA were built inside the MCM channel. The double helix bends by 10° as it enters the N-terminal dimerization domain. c Overlay between the 6.9 Å resolution, DNA-occupied structure and the higher-resolution ensemble protein structure fails to highlight detectable conformational changes in MCM upon DNA binding. A composite resolution structure can therefore be built integrating the atomic models derived from the lower resolution DNA and higher-resolution protein structures. d Detailed analysis of the MCM pore loops engaging the Watson and Crick strands. OB, oligosaccharide/oligonucleotide fold. PS1, Pre-sensor 1 hairpin. Zf zinc-finger hairpin, H2i helix-2-insert

Fig. 5

A model for origin DNA opening: MCM-to-CMG transition causes DNA melting inside the helicase channel. a, b Duplex DNA encircled by the N-terminal MCM Zinc fingers becomes underwound as it transitions from MCM to CMG. Concomitantly, the ATPase-captured strand becomes stretched and untwisted inside the helicase pore. c MCM pore loop elements interacting with duplex DNA. Notably, ATPase pore loops Mcm6 PS1 h and Mcm3/5 h2i hairpins engage both the Watson and Crick strands. d MCM pore loops in the CMG touch the stretched and untwisted DNA. Mcm6 PS1 hairpin moves upwards by 12 Å. Mcm7 OB hairpin moves downwards by 9 Å and pushes against the DNA junction. Mcm4 remains fixed in proximity to the DNA junction. Mcm3/5 h2i hairpins stabilize the stretched configuration of the ATPase-captured strand

Fig. 6

Model for replication initiation. MCM is loaded onto origin DNA in a process that requires ATP hydrolysis by MCM. In the loaded double hexamer, duplex DNA runs through the entire length of the central channel. Upon DDK phosphorylation the N-terminal tails of Mcm4-6 become more stable, probably forming a landing platform for the Cdc45-recruiting factor, Sld3/Sld7. Upon binding of GINS and Cdc45, MCM melts the double helix. The directionality of CMG movement after replication fork establishment remains a matter of debate