Structural and mechanistic insights into Mcm2-7 double-hexamer assembly and function

Abstract

Eukaryotic cells license each DNA replication origin during G1 phase by assembling a prereplication complex that contains a Mcm2-7 (minichromosome maintenance proteins 2-7) double hexamer. During S phase, each Mcm2-7 hexamer forms the core of a replicative DNA helicase. However, the mechanisms of origin licensing and helicase activation are poorly understood. The helicase loaders ORC-Cdc6 function to recruit a single Cdt1-Mcm2-7 heptamer to replication origins prior to Cdt1 release and ORC-Cdc6-Mcm2-7 complex formation, but how the second Mcm2-7 hexamer is recruited to promote double-hexamer formation is not well understood. Here, structural evidence for intermediates consisting of an ORC-Cdc6-Mcm2-7 complex and an ORC-Cdc6-Mcm2-7-Mcm2-7 complex are reported, which together provide new insights into DNA licensing. Detailed structural analysis of the loaded Mcm2-7 double-hexamer complex demonstrates that the two hexamers are interlocked and misaligned along the DNA axis and lack ATP hydrolysis activity that is essential for DNA helicase activity. Moreover, we show that the head-to-head juxtaposition of the Mcm2-7 double hexamer generates a new protein interaction surface that creates a multisubunit-binding site for an S-phase protein kinase that is known to activate DNA replication. The data suggest how the double hexamer is assembled and how helicase activity is regulated during DNA licensing, with implications for cell cycle control of DNA replication and genome stability.

Keywords: DNA replication initiation; electron microscopy; origin recognition complex; prereplication complex; replicative helicase.

Figure 1.

The multistep pre-RC assembly process and EM visualization of intermediates during recruitment of the first Mcm2–7 hexamer by ORC–Cdc6. (A) A sketch for loading of the Mcm2–7 double hexamer highlighting three unresolved issues as shown by three question marks: (1) Does ATP hydrolysis and Cdt1 release from OCCM lead to a large gap between ORC-Cdc6 and Mcm2–7 that may allow ORC–Cdc6 to recruit a second Mcm2–7 hexamer (step 3)? (2) How is the second Mcm2–7 hexamer recruited (step 4)? (3) How are the two Mcm2–7 hexamers arranged in the double hexamer (step 5)? (B) The OCM structure compared with the OCCM structure. (Top panel) 2D class averages of cryo-EM (top row) and negative stain images (bottom row) of OCCM formed in the presence of ATP-γS. Red arrows point to the Cdt1 density identified previously. (Bottom panel) 2D class averages of OCM in the presence of ATP at a 7-min reaction time by cryo-EM (top row) and negative stain EM (bottom row). Blue arrows point to the regions where Cdt1 is missing. In both panels, a selected 2D average is enlarged and overlaid with sketches of the proposed ORC–Cdc6 region in orange and the Mcm2–7 region in purple. Bar, 15 nm.

Figure 2.

A novel loading intermediate containing one OCM and one Mcm2–7 hexamer. (A) Four class-averaged, negative stain EM images of the novel five-tiered Mcm2–7 loading intermediate. Particles were selected from a sample that was incubated in 3 mM ATP for 7 min followed by 0.1% glutaraldehyde cross-linking. (B) Selected reference-free 2D averages of a Mcm2–7 double hexamer found in the same sample as in A. (C) The OCMM raw particle images found in the 7-min reaction sample without glutaraldehyde cross-linking. (D) The OCMM is an on-pathway intermediate during the loading of the Mcm2–7 double hexamer. The question mark denotes the possible existence of additional intermediates upstream of OCMM.

Figure 3.

3D reconstructions of six MBP-inserted Mcm2–7 double hexamers. (A) Two selected reference-free class averages of the double hexamer with MBP inserted to the N-terminal region of Mcm2 (MBP-M2), Mcm3 (MBP-M3), Mcm6 (MBP-M6), and Mcm7 (MBP-M7) and the C-terminal region of Mcm2 (M2-MBP) and Mcm5 (M5-MBP). Blue asterisks mark the extra and peripheral MBP densities. Box size is 36 nm. (B) Each column shows the top, front, and side views of the six MBP-fused Mcm2–7 double hexamers. The MBP density is colored blue in the 3D map. The bottom panels show horizontal sections of the 3D maps at the inserted MBP positions as indicated by dashed lines. Blue asterisks mark the MBP densities in the sections. Box size in the bottom panel is 24 nm. (C) The Mcm subunit arrangement in the lower hexamer as viewed from the N termini (left) and in the upper hexamer viewed from the C termini (right). (D) NTD and CTD assignment of each Mcm subunit in the top hexamer of the double hexamer as viewed from the side. The NTDs and the CTDs are staggered such that each Mcm subunit is tilted in the hexamer, and the double hexamer is twisted. The Mcm protein domains labeled with red numbers in C and D are positively identified in this study, and the domains in blue numbers are inferred from the known order of subunits in the hexamer.

Figure 4.

The architecture of the Mcm2–7 double hexamer. (A) Four side views of the 3D map of the wild-type Mcm2–7 double hexamer consecutively rotated 90° around a vertical axis. The six Mcm proteins are colored differently and labeled correspondingly. A pair of blue lines trace the helical arrangement of the NTDs (red asterisks) of Mcm2, Mcm6, and Mcm4 (back side view). The black ovals in the front and back views indicate the position of the twofold axis. (B) Top and bottom views of the Mcm2–7 CTD ring. A density that is connected to the CTD of Mcm3 and located near the hexamer center is tentatively assigned as the CTE of Mcm3. (C) Top and bottom views of the Mcm2–7 NTD ring. Overlapping the two views by translation generates the interface between two hexamers of the Mcm2–7 double hexamer. Mcm2–Mcm5 in one hexamer is approximately aligned with Mcm4–Mcm6 in the other.

Figure 5.

Functional implications of the Mcm2–7 double-hexamer structure. (A) There are two types of tilt found in the double-hexamer EM structure. The first is the end-to-end stacking of the two hexamers that is staggered by ∼2 nm and tilted by ∼5° off the vertical cylinder axis. The second is a ∼30° tilt of individual Mcm subunits in each hexamer with respect to the vertical axis. (B) The ATP hydrolysis rates were determined for the Mcm2–7 helicase in single-hexamer (H) and double-hexamer (DH) conformations. The single hexamer exhibits a robust ATPase activity that is strongly reduced in the double-hexamer configuration. (C, left) A speculative sketch illustrating that the arginine finger (R) of one Mcm subunit in the hexamer is properly positioned toward the Walker A and B motifs (A/B) of a neighbor protein for ATPase activity. (Right) Subunit tilt in the double hexamer misaligns the arginine finger (R) with respect to the A/B motifs and abolishes the ATPase activity. (D) The backside view of the double-hexamer structure shows that the Mcm2/5 interface in the upper hexamer is nearly opposite to the Mcm2/5 interface in the lower hexamer. The NTD of Mcm4 (M4N) wedges against the potential Mcm2/5 gate. (E) A sketch illustrating that the potential Mcm2/5 is unconstrained in the single hexamer (shown at left) but is constrained or locked by structural features such as the Mcm4 NTD of the opposing hexamer in the double hexamer. (F) A model for DDK interaction with the Mcm2–7 double hexamer but not with the single hexamer. The heterodimeric DDK requires a bipartite binding site (NTDs of Mcm2 and Mcm4) that is not available in the single hexamer but is available in the double hexamer. The double hexamer is in the right side view as in Figure 4A. (G) DDK activity on the purified single versus double Mcm2–7 hexamers. The top panels are by silver staining, and the bottom panel is by ³²P autoradiography.

Figure 6.

EM of pre-RC intermediates suggests possible helicase loading and initial DNA-melting mechanisms. Steps 1–5 describe the proposed model for the eukaryotic pre-RC formation. Each of the five steps as drawn is based on an experimentally captured Mcm2–7 loading intermediate. ORC–Cdc6 on origin DNA in step 1 is the activated platform for loading of Cdt1–Mcm2–7. Formation of OCCM as shown in step 2 represents the recruitment of the first hexamer. ATP hydrolysis in step 3 leads to the release of Cdt1 and formation of OCM. Formation of the OCMM in step 4 completes the recruitment of the second hexamer. The question mark in step 4 indicates a possible short-lived intermediate not captured in the present study. In step 5, ATP hydrolysis by ORC–Cdc6 leads to maturation of the double-hexamer structure on DNA and its separation from ORC–Cdc6. Mcm subunits are colored purple in the single hexamer but are colored orange in the double hexamer to highlight the subunit tilt. The two hexamers in the OCMM are also colored orange (step 4) because their structure is highly similar to the final double hexamer. The OCM, OCMM, and double hexamer in steps 3, 4, and 5 are characterized in this study. During the transition from step 5 to step 6, a possible untwisting of the right-hand-tilted Mcm proteins in the double hexamer may locally unwind the dsDNA. Further modification by DDK and CDK and binding to Cdc45 and GINS lead to the separation of the double hexamer and formation of two CMG complexes, each encircling ssDNA (step 6).