CryoEM structures of human CMG-ATPγS-DNA and CMG-AND-1 complexes

Abstract

DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPγS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPγS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one β-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins.

Figure 1.

Structure of the human CMG–ATPγS–DNA DNA helicase. Top (A) and side (B) views of a ribbon model of the atomic structure of CMG–ATPγS–DNA. The CMG components are labelled in the top view; the N- and C-tier of the CMG are highlighted in the side view. The MCM2–7 subunits are coloured according to MCM chain: MCM2 is pink, MCM3 cyan, MCM4 green, MCM5 yellow, MCM6 orange and MCM7 blue, following the colour scheme of Eickhoff and colleagues (19); Cdc45 and GINS are coloured grey and the single-stranded (ss) DNA in white. To the top left of each CMG–ATPγS–DNA view, the 3.3 Å cryoEM map in the same orientation is also shown. The regions of the map corresponding to different CMG subunits are coloured as the ribbon model.

Figure 2.

Protein-DNA interactions in the human CMG–ATPγS–DNA complex. MCM chains are coloured coded as in Figure 1. (A) Side view of the CMG–ATPγS–DNA complex, highlighting the position of ssDNA within the MCM C-tier. The MCM subunits are shown as transparent molecular surfaces and the DNA is drawn as a dark grey ribbon. Cdc45 and GINS have been omitted for clarity. (B) Schematic drawing illustrating the DNA footprint and ATP status of each MCM subunit. (C) The staircase configuration of the H2I and PS1 DNA-binding loops around the ssDNA spiral, from MCM2 (pink) at the top (5′-end of the DNA) to MCM5 (yellow) at the bottom (3′-end).

Figure 3.

Atomic details of the CMG - DNA interface. The central panel shows a schematic drawing of the 11-nucleotide ssDNA and the MCM residues that interact with DNA in the H2I and PS1 loops of each subunit. In the six surrounding boxes, close-up views of the protein - DNA interface for each MCM subunits are shown. MCM chains are color-coded as in Figure 1.

Figure 4.

ATPγS binding in the CMG–ATPγS–DNA complex. In the middle-right panel, the MCM C-tier is shown in molecular surface representation, clipped from the N-tier end, to reveal the nucleotide bound at each interface of the MCM C-tier. The cartoon drawing above the panel recapitulates the ATP status of MCM2–7 (AGS stands for ATPγS). Details of nucleotide binding are provided in the oval panels for each of the five nucleotide-bound interfaces. MCM chains are colour-coded as in Figure 1.

Figure 5.

Conformational coupling of ATP status and DNA-binding loops. (A) Spacefill representation of the MCM C-tier, colour-coded according to ATP status. (B) Ribbon representation of MCM4 and DNA, showing the reciprocal position of the ATPγS moiety and the H2I and PS1 DNA-binding loops. (C) Superposition of MCM ATPase domains, highlighting the relative position of their DNA-binding loops (MCM2 is omitted, see Supplementary Figure S11). The H2I α_N helix of MCM5 is disordered and drawn as a dotted line. The MCM ATPase domains are coloured according to ATP status, as in panel A.

Figure 6.

Structure of human CMG bound to AND-1 (CMGA). Top and side views of the atomic model of CMGA are shown in ribbon drawing. The MCM ring is coloured in grey, Cdc45 and GINS in pink and AND-1 in yellow. At the top-left of each ribbon drawing is reported a view of the cryoEM map in the same orientation, coloured accordingly. A schematic drawing of the domain structure of AND-1, consisting of N-terminal WD40 domain, a central SepB domain and a C-terminal HMG box domain, is shown in the top right corner.

Figure 7.

The CMG - AND-1 interface in CMGA. The middle panel shows transparent molecular surfaces for GINS (light pink), Cdc45 (darker pink) and AND-1 (light and dark yellow). The three β-propeller domains of the AND-1 SepB trimerisation region are shown as ribbons: the β-propeller that contacts the CMG is coloured from blue (N-end) to red (C-end). The six blades of the propeller are numbered 1 (N-end) to 6 (C-end). To the left is a view of the cryoEM map for the CMGA, with ribbon models of AND-1 (yellow) and Cdc45 (pink) fitted in the map. Above and below the central panels are ribbon drawings showing the interface of AND-1’s SepB β-propeller domain with the B-box of Psf2 (top) and with Cdc45 (bottom). Cdc45 and Psf2 are in pink, with their AND-1-binding regions highlighted in magenta, whereas AND-1’s SepB β-propeller is coloured from blue (N-end) to red (C-end), as in the central panel.