Sld3CBD–Cdc45 complex structure

(A) Structure of the Sld3CBD–Cdc45 complex. Dotted circles B, C, and D mark three binding sites, corresponding to the bottom panel. Proteins Sld3CBD and Cdc45 are colored in green and magenta, respectively. The DHHA1 of Cdc45 is labeled and colored in dark magenta. (B) Binding site on the α8CTP of Sld3CBD. In (C) and (D), two binding sites involving hydrophobic and hydrogen-bond interactions on the α9 of Sld3CBD, respectively. The interacted residues are depicted by sticks and labelled. The black dotted lines show hydrogen bonds.

Mutation analysis of interacted residues

(A) In vitro binding analysis was checked using SDS-PAGE after Ni-affinity chromatography extraction of co-overexpressed Cdc45 with each Sld3 mutant. Sld3 was tagged by His-tag to bind to the column. The labels M, W, Y, 3E, 3S, and 2R are explained on the right. (B) In vitro binding analysis was checked using SDS-PAGE after Ni-affinity chromatography extraction of co-overexpressed Sld3 with each Cdc45 mutant. Sld3 was tagged by His-tag to bind to the column. The labels M, W, A, IIIE, IIE, IIIS, and IIS are explained on the right. (C) In vivo cell growth analysis of yeast cells carrying sld3 mutations. The yeast YYK13 cells carrying SLD3 or its mutant plasmids were streaked onto SD and FOA plates and then incubated at 298 K for 3 days. YYK13 yeast is a mutant lacking the SLD3 gene with added SLD3/sld3 mutant gene (YCplac22 plasmid containing SLD3 or sld3 mutant) that grew on SD and FOA plates. The empty plasmids were used as negative control (NC). Mutations in Sld3-Y, Sld3-3E, Sld3-3S, and Sld3-2R are the same as those in A.

Ribbon models of complexes in dimer form and particle analysis

(A) Sld3CBD–Cdc45–MCM–dsDNA complex. Mcm2, 5, 4, and 6 subunits are colored in cyan, blue, marine, and light blue, respectively. Subunits Mcm3 and Mcm7 are colored in gray. Green and pink are used to color Sld3CBD and Cdc45, respectively. dsDNA is represented by a dark-orange stick. (B) Sld3CBD–Sld7–Cdc45 dimer before associating with the MCM DH. Sld3 and Cdc45 are shown in the same color as they are in A, while Sld7 is colored in orange. The two phosphorylated Sld3 residues are depicted as yellow balls. Particle analysis of Sld7–Sld3ΔC– Cdc45 through dynamic light scattering is shown on the bottom panel. The average peak size of the particle size distribution of the Sld7–Sld3ΔC–Cdc45 complex was estimated to be 23.5 Å in diameter. The measurement was carried out independently four times (Supplementary Figure 9).

(C) SCMG–dsDNA complex. GINS is shown in yellow and the remainder are colored identically to those in A and B.

Electrophoresis mobility shift assay (EMSA) of ssDNA binding to Sld3 and its complexes with Sld7 and Cdc45

(A) Schematic of ssARS1-1–ssARS1-6. The important elements of A (ARS consensus sequence), B1, B2, and B3 for unwinding are marked [33]. We divided ssARS1-2 and ssARS1-5 fragments (blue squares) into 40-base lengths for EMSA. (B) ssDNAs were visualized using SYBR safe on polyacrylamide gels. In the presence of ssDNA fragments, Sld3CBD, Sld3CBD– Cdc45, Sld7–Sld3ΔC–Cdc45 and Sld7–Sld3ΔC were incubated with molecular mass-related concentrations. The molecular ratio of ssDNA to protein in lanes 1, 2, 3, 4, and 5 was 1:0, 1:1, 1:2, 1:4, and 0:1, respectively. The controls for ssDNA and protein are lanes 1 and 5, respectively. The disappearance of the ssDNA band in lanes 2–5 indicates that the protein (Sld3CBD, Sld7–Sld3ΔC and Sld7–Sld3ΔC–Cdc45) binds with high affinity.

Proposal for CMG formation with Sld7–Sld3

(A) Phosphorylation of Mcm2,4,6 by DDK after MCM DH was loaded on dsDNA at the replication origin. (B) Cdcd45 recruitment to MCM DH by Cdc45–Sld3–[Sld7]2–Sld3– Cdcd45. (C) After CDK-mediated phosphorylation of Sld3CTD in Cdc45–MCM, Dpb11– Sld2 recruits GINS and polε to Sld7–Sld3-Cdc45–MCM to form an active helicase CMG.

(D) Unwinding of dsDNA by CMG with MCM DH separation and MCM ring opening. Sld3 and other factors are released upon binding to ssDNA. (E) Each CMG unwinds the dsDNA in two directions, initiating DNA replication.