Structural insights into the Cdt1-mediated MCM2-7 chromatin loading

Abstract

Initiation of DNA replication in eukaryotes is exquisitely regulated to ensure that DNA replication occurs exactly once in each cell division. A conserved and essential step for the initiation of eukaryotic DNA replication is the loading of the mini-chromosome maintenance 2-7 (MCM2-7) helicase onto chromatin at replication origins by Cdt1. To elucidate the molecular mechanism of this event, we determined the structure of the human Cdt1-Mcm6 binding domains, the Cdt1(410-440)/MCM6(708-821) complex by NMR. Our structural and site-directed mutagenesis studies showed that charge complementarity is a key determinant for the specific interaction between Cdt1 and Mcm2-7. When this interaction was interrupted by alanine substitutions of the conserved interacting residues, the corresponding yeast Cdt1 and Mcm6 mutants were defective in DNA replication and the chromatin loading of Mcm2, resulting in cell death. Having shown that Cdt1 and Mcm6 interact through their C-termini, and knowing that Cdt1 is tethered to Orc6 during the loading of MCM2-7, our results suggest that the MCM2-7 hexamer is loaded with its C terminal end facing the ORC complex. These results provide a structural basis for the Cdt1-mediated MCM2-7 chromatin loading.

Figure 1.

Solution structure of hMBD of human Cdt1 and the Mcm6-Cdt1 complex. (a) The left panel shows the backbone superposition of the 20 lowest-energy NMR structures of hMBD. The α-helix is colored red. N-terminal and C-terminal ends are indicated as N and C. The right panel shows a ribbon representation of the same structure of hMBD using the coordinates of the lowest energy structure. (b) Chemical shift perturbations in the presence of hCBD are colored onto the structure of hMBD in the surface representation. Residues with chemical shift perturbations ranging from 0.00 to 0.08 ppm are colored in green while residues with chemical shift perturbations larger than 0.08 ppm are shown in red and residues disappeared in HSQC spectrum are in blue. (c) Backbone superposition of the 19 lowest-energy NMR structures. Secondary structural elements of hCBD are color-coded: α-helices (red), β-strands (green), and loops (gray). hMBD is shown in blue. N-terminal and C-terminal ends are indicated as N and C. (d) Ribbon diagram of the complex using the coordinates of the lowest energy structure. (e) Expanded view of the complex binding surface. Residues having intermolecular NOEs are shown in sticks, yellow for hCBD and green for hMBD. The backbones of hCBD and hMBD are colored gray and blue respectively. (f) Surface representation of hCBD colored by residue type: red, acidic; blue, basic; yellow, hydrophobic; gray, non-interacting. hMBD is indicated as in (e) except that the backbone is in purple.

Figure 2.

NMR studies of the interaction between hMBD and hCBD. (a) Overlays of ¹H-¹⁵N HSQC spectra of ¹⁵N-labeled hMBD in free form (black) titrated with hCBD at a molar ratio of 1:3 (blue), 1:6 (red). The arrows point to the shifted positions (black to red) of the amide proton resonance. (b) Chemical shift differences between the free-form and hCBD saturated hMBD. NMR titration experiments show an intermediate exchange kinetics indicating a relatively strong binding between hCBD and hMBD.

Figure 3.

Intermolecular NOEs between hCBD and hMBD. The left two strips of a 3D F₁ ¹³C,¹⁵N-filtered, F₂ ¹³C-edited NOESY-HSQC spectrum (150 ms mixing time) of a sample containing ¹³C,¹⁵N-labeled hCBD and unlabeled hMBD showing NOEs from the hMBD (I426, L422, the ambiguous assignment is colored by blue) to the hCBD (K770, I767). The right strip of the same experiment of a sample containing ¹³C, ¹⁵N-labeled hMBD and unlabeled hCBD showing NOEs from the hCBD (L766, K770) to the hMBD (L422). CD1: the ε1 carbon atom of the Isoleucine methyl group; CD#: degenerate pairs of Leucine methyl carbon atoms; CE: the ε carbon of the Lysine; HA: the α proton attached to α carbon; QG: degenerate pairs of γ methylene protons; HG: the γ proton of Leucine; QG1: degenerate pairs of Isoleucine γ1 methylene protons; QD: degenerate pairs of δ methylene protons; QE: degenerate pairs of ε methylene protons; QD1: degenerate pairs of Isoleucine δ1 methyl protons; QQD: degenerate pairs of Leucine methyl protons.

Figure 4.

Disruption of the Cdt1–Mcm6 interaction by mutations on their interacting surfaces impaired pre-RC formation and DNA replication. (a) Wild-type and mutants of hMBD were pulled down by GST-tagged wild-type hCBD and visualized by Coomassie Blue staining. (b)Chromatin loading of Mcm2 (S. cerevisiae) is interrupted by the Mcm6-5A mutant (Supplementary Material). (c) Chromatin loading of Mcm2 (S. cerevisiae) is interrupted by the Cdt1-3A mutant (Supplementary Data). (d) Mcm6-5A cells arrest in S-phase with large buds (S. cerevisiae) (Supplementary Data). Flow cytometry was performed for the cell samples taken at the indicated time points. % Bud. percentage of budding cells. (e) Cdt1-3A cells arrest in S-phase as in (d).

Figure 5.

Alignments of the Cdt1 and Mcm6 sequences from different organisms. (a) Multiple sequence alignment of a representative set of Mcm6 proteins: Q14566, H. sapiens; P97311, Mouse; BAF94254, Rattus norvegicus; Q5FWY4, XENLA; Q9V461, D. melanogaster; P4973, S. pombe; P53091, S. cerevisiae. The dashes indicate the positions of gaps in eukaryotic sequences. Secondary structural elements at the top of the alignment are indicated with color coding as in Figure 1a and b. (b) Multiple sequence alignment of a representative set of the C-terminal regions of Cdt1: Q9H211, H. sapiens; Q8R4E9, Mouse; D3ZKD4, Rattus norvegicus; Q9I9A7, XENLA; Q7JVY2; D. melanogaster; P40382, S. pombe; P47112, S. cerevisiae. The sequence alignment was produced with ClustalX(34). Blue arrows indicate the amino acids that greatly reduce the interaction between Mcm6 and Cdt1 upon Ala substitution. Residues are gray-scaled based on percentage identity.

Figure 6.

Proposed model for the Cdt1/MCM2-7 recruitment and MCM hexmer loading events. The Cdt1 functions as the bridge to bring Orc6 and MCM2–7 together to form pre-RC. The C-terminal Mcm6-binding domain of Cdt1 tethers MCM2–7, while the N-terminal Orc6-binding domain recruits Cdt1/MCM2-7 to ORC complex.