Sld3CBD–Cdc45 structural insights into Cdc45 recruitment for CMG complex formation during DNA replication
- Graduate School of Life Science, Hokkaido University, Japan
- Faculty of Advanced Life Science, Hokkaido University, Japan
- National Institute of Genetics, Japan
Figures
Figure 1 with 6 supplements
Sld3CBD–Cdc45 complex structure.
(A) Structure of the Sld3CBD–Cdc45 complex. Sld3CBD and Cdc45 are colored in green and magenta, respectively. Cdc45-binding parts α8CTP and α9 are labelled and colored in cyan. The DHHA1 of Cdc45 is labelled and colored in dark magenta. (B) Binding part of Sld3CBD-Cdc45 in different viewing. Dotted squares C, D, and E mark three binding sites, corresponding to the bottom panel. (C) Binding site on the α8CTP of Sld3CBD. (C) (D) Two binding sites involving hydrophobic and hydrogen-bond interactions on the α9 of Sld3CBD, respectively. The interacting residues are depicted by sticks and labelled. The black dotted lines show hydrogen bonds.
Figure 1—figure supplement 1
Formation of the Cdc45–MCM–GINS (CMG) complex after MCM double hexamer (DH) bound to the replication initiation site.
(A) MCM DH binds to the replication origin and then is phosphorylated by DDK. (B) Sld3 and Sld7 recruit Cdc45 to the MCM DH complex. (C) After CDK phosphorylates Sld3, Dpb11 and Sld2 recruit GINS to MCM DH to form the active helicase CMG complex. (D) Finally, factors other than CMG (Cdc45–MCM–GINS) are dissociated, and double-stranded DNA (dsDNA) is unwound to ssDNA in preparation for initiating replication.
Figure 1—figure supplement 2
Purification of the Sld3CBD–Cdc45 and Sld7–Sld3ΔC–Cdc45 complexes.
Size-exclusion chromatography (SEC) of ScSld3CBD–Cdc45 (A) and KmSld7–Sld3ΔC–Cdc45 (B). SDS-PAGE was performed to examine the purity of each sample in the SEC plots. Lane M shows the molecular weight markers labelled in kDa. At the upper left, we collected the principal peak in the SEC as Sld3CBD–Cdc45, which was used in other experiments. According to the bottom-right image, we collected the first half of the marked peak in SEC (B left) as purified Sld7–Sld3ΔC–Cdc45. A standards kit was measured using Superdex 200 16/60 column to check the elution volume shift of Sld7–Sld3ΔC–Cdc45. The molecular weight at the peak elution position of Sld7-Sld3ΔC-Cdc45 was estimated to be 429 kDa.
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Figure 1—figure supplement 2—source data 1
PDF file containing original SDS-PAGE for Figure 1—figure supplement 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig1-figsupp2-data1-v1.pdf
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Figure 1—figure supplement 2—source data 2
Original files for SDS-PAGE displayed in Figure 1—figure supplement 2.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig1-figsupp2-data2-v1.zip
Figure 1—figure supplement 3
The topology diagram of Sld3CBD-Cdc45.
The topology diagram of Sld3CBD and Cdc45 in the Sld3CBD-Cdc45 complex structure. α helix and β sheet were shown by the square and the arrow, respectively. The black line and dotted line represent the loop and disorder regions, respectively.
Figure 1—figure supplement 4
Structural Comparison of Sld3CBD and Cdc45.
(A) Sld3CBD in the Sld3CBD–Cdc45 complex (left) and the isolated structure of Sld3CBD (3WI3) (right). The black circle indicates a long helix α8CTP that was only visible in the Sld3CBD–Cdc45 complex with an average B factor of 45 A2 for the main chain. (B) Cdc45 in Sld3CBD–Cdc45 (left; magenta), CMG (3JC6) (center; yellow-green), and isolated HuCdc45 structure (5DGO) (right; cyan). In Sld3CBD–Cdc45, the black circle indicates a long helix that was partly disordered. (C) Superposition of Cdc45 in Sld3CBD–Cdc45 by aligning Cdc45NTD (~K517) to Cdc45 in the Cdc45–MCM–GINS (CMG) complex and huCdc45, respectively (upper panel); colors are the same as those in (B). The black dotted circles indicate conformationally changed DHHA1 domains (magnified below the images). Significant changes in the α19 and downstream β-sheets in the DHHA1 domain are highlighted by the black arrow and circle, respectively. The parts with no significant conformational changes are colored gray, and the colors of the other parts are the same as those in (B).
Figure 1—figure supplement 5
Sequence alignment of Sld3/Treslin domain with structural elements.
Sequence alignment of the Sld3/Treslin domain (Cdc45-binding domain: CBD) with structural elements from fungal Sld3 (S. cerevisiae, K. marxianus, and Ashbya gossypii) and vertebrate Treslin/Ticrr (Homo sapiens, Mus musculus, and Xenopus laevis). The sequences are as follows: Sld3_SACCE, S. cerevisiae; Sld3_KLUMA, K. marxianus; Sld3_ASHGO, Ashbya gossypii; Ticrr_HUMAN, Homo sapiens; Ticrr_MOUSE, Mus musculus; and Ticrr_XENLA, Xenopus laevis. CLUSTAL W (https://www.genome.jp/tools-bin/clustalw) was used to create an initial alignment, which was modified based on the 3D structure. Structural elements of TICRR_HUMAN were predicted using PSIPRED 4.0 (http://bioinf.cs.ucl.ac.uk/psipred/). * conserved sequence;: and. conserved change. Amino acids are marked and colored red, green, and blue. The secondary structures of Sld3CBD (in Sld3CBD–Cdc45) and predicted TICRR_HUMAN are shown above and below the alignment, respectively. Dashed lines in the secondary structure indicate disordered regions. The residue numbers for SLD3_SACCE are indicated above the alignment. The numbers in parentheses beside the sequences indicate the number of residues in each protein. The mutation sites used in this study are indicated by black arrows.
Figure 1—figure supplement 6
Sequence alignment of Cdc45s with structural elements.
Sequence alignment of Cdc45s with structural elements from fungi (S. cerevisiae, K. marxianus, and Ashbya gossypii) and vertebrates (Homo sapiens, Mus musculus, and Xenopus laevis). The sequences are indicated as Cdc45_SACCE, S. cerevisiae; Cdc45_KLUMA, K. marxianus; Cdc45_ASHGO, Ashbya gossypii; Cdc45_HUMAN, Homo sapiens; Cdc45_MOUSE, Mus musculus; and Cdc45_XENLA, Xenopus laevis. CLUSTAL W (https://www.genome.jp/tools-bin/clustalw) was used to create an initial alignment, which was modified based on the 3D structure. * conserved sequence;: and. conserved change. Amino acids are marked and colored red, green, and blue. Secondary structures of Cdc45 in Sld3CBD–Cdc45 and HuCdc45 (PDBID: 5DGO) are indicated above and below the alignment, respectively. Dashed lines in the secondary structure indicate disordered regions. The residue numbers for Cdc45_SACCE are shown above the alignment. The numbers in parentheses beside the sequences indicate the number of residues in each protein. Black arrows indicate the mutation sites used in this study. A black frame shows the domain DHHA1.
Figure 2 with 3 supplements
Mutation analysis of interacting 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 a negative control (NC). Mutations in Sld3-Y, Sld3-3E, Sld3-3S, and Sld3-2R are the same as those in A.
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Figure 2—source data 1
PDF file containing original SDS-PAGE for Figure2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig2-data1-v1.pdf
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Figure 2—source data 2
Original files for SDS-PAGE displayed in Figure2.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig2-data2-v1.zip
Figure 2—figure supplement 1
In vivo mutation analysis of Cdc45 using mutant cells.
SLD3 on the high-copy YEplac195 plasmid and SLD7 on the high-copy YEplac112 plasmid were introduced into mutant cells bearing a mutation that replaced Cdc45 Ser242 with proline on the Sld3 binding surface (Cdc45S242P). The transformants were streaked onto yeast extract–peptone–dextrose plates and individually incubated for 3 days at 25°C and 32°C. Blank plasmids YEplac195 and YEplac122 were used as negative controls (NC). CDC45 was introduced into the YEplac195 plasmid as a positive control. Cell growth was suppressed by Cdc45S242P mutation at 32°C.
Figure 2—figure supplement 2
SDS-PAGE analysis and circular dichroism spectra of Sld3 mutants.
Structural elements of Sld3-3S, Sld3-3E, Sld3-2R, and Sld3-Y (Sld3-3S: I352S/I355S/L356S, Sld3-3E: I352E/I355E/L356E, Sld3-2R: D344R/D348R, and Sld3-Y: I352Y) were analyzed through circular dichroism. While the mutants of Sld3CBD existed alone, we prepared WT Sld3CBD in a complex with Cdc45 and calculated the elements of secondary structure from the crystal structure of Sld3CBD–Cdc45. All variants appeared to maintain the same structural elements as wild-type Sld3CBD–Cdc45, as indicated in the table. The concentration of samples was controlled to the same level for CD measurement.
Figure 2—figure supplement 3
Mutation analysis of Cdc45.
(A) Binding site of Cdc45 to Sld3CBD, GINS, and MCM. Two Cdc45 sites involved in binding to MCM (Cdc45 G367) and GINS (Cdc45 W481) are colored in red. (B) In vitro binding analysis was checked using SDS-PAGE after Ni-affinity chromatography extraction of co-overexpressed Sld3CBD with each Cdc45 mutant. Sld3CBD with His-tag bound to the column. The labels M, W, R, and D are explained on the right.
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Figure 2—figure supplement 3—source data 1
PDF file containing original SDS-PAGE for Figure 2—figure supplement 3, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig2-figsupp3-data1-v1.pdf
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Figure 2—figure supplement 3—source data 2
Original files for SDS-PAGE displayed in Figure 2—figure supplement 3.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig2-figsupp3-data2-v1.zip
Figure 3 with 3 supplements
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 232 Å in diameter. The measurement was carried out independently four times (Figure 3—figure supplement 1). (C) SCMG–dsDNA complex. GINS is shown in yellow, and the remainder are colored identically to those in A and B.
Figure 3—figure supplement 1
Dynamic light scattering (DLS) of Sld7–Sld3ΔC–Cdc45.
Four samples of the Sld7–Sld3ΔC–Cdc45 complex were measured by DLS. Each sample was overexpressed independently and purified through size-exclusion chromatography. For data analysis, each sample was measured three times. The particle size was estimated to be 232 Å in average of peak size from four samples. The pure buffer was measured as a background control and 10 μM lysozyme was measured as a standard control, shown by orange and blue curves, respectively.
Figure 3—figure supplement 2
Size-exclusion chromatography (SEC)-small-angle X-ray scattering (SAXS) analysis of Sld7–Sld3ΔC–Cdc45.
SEC-SAXS measurements of Sld7–Sld3ΔC–Cdc45 complex were performed using a beam line BL-10C at the Photon Factory (Tsukuba, Japan). SEC-SAXS data were collected under camera length 2 M, wavelength 1.5 Å, and 20℃ with a program Seral Analyzer (upper panel) (Yonezawa et al., 2019). A program SAngler was used to analyze the SEC-SAXS data (Shimizu et al., 1741). The Guinier plot (left) and Kratky plot (right) are shown in the lower panel. Rg and Dmax were estimated around 85 and 345 Å, respectively. Ascent side: the SAXS data collated from the left side of the SEC-plot peak. Peak: the SAXS data collected from a peak point of the SEC plot.
Figure 3—figure supplement 3
DHHA1 domains of Cdc45s.
The DHHA1 domains of Cdc45 in the Cdc45–MCM–GINS (CMG) complex (PDB ID: 3JC6) (A) and the SCMG–double-stranded DNA (dsDNA) model (B), are shown. DHHA1s are colored in red and around the black circles. Labelled 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 were used to indicate Sld3CBD and Cdc45, respectively. GINS is shown in yellow and dsDNA is presented as a dark orange stick. The contact area between DHHA1 and Mcm2 is magnified in the bottom panel of the figure. The black dotted circles mark the contact between DHHA1 and Mcm2CTD in CMG complex (A) and SCMG–dsDNA model (B), respectively. The predicted closed residues between Cdc45 and Mcm2 in the SCMG–dsDNA model are labelled.
Figure 4 with 5 supplements
Electrophoresis mobility shift assay (EMSA) of single-stranded DNA (ssDNA) binding to Sld3 and its complexes with Sld7 and Cdc45.
(A) Schematic of ssARS1-1–ssARS1-6. ARS1 is identified as an origin of DNA replication and divided into three 80 bp segments: ARS1-12, ARS1-34, and ARS1-56. These double-stranded DNA (dsDNA) segments could unwind into six single-stranded DNA fragments with 80 nt length: ssARS1-1, 2, 3, 4, 5, and 6. The important elements of A (autonomously replicating sequence, ARS consensus sequence), B1, B2, and B3 for unwinding are marked (Newlon and Theis, 1993). We divided ssARS1-2 and ssARS1-5 fragments (blue squares) into 40-base lengths for electrophoretic mobility shift assay (EMSA). (B) ssDNAs were visualized using Fast Blast DNA stain 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, 1:0.5, 1:1, 1:2, and 0:1, respectively. The controls for ssDNA and protein are lanes 1 and 5, respectively. No binding ssDNA group (ssARS1-3_1) is shown at the bottom as a negative control (NC). The overall views of the EMSA results are shown in Figure 4—figure supplement 2. (C) The integral grayscale of the ssDNA bands was calculated and compared to the average of the ssDNA control band to determine the residual levels, showing differences in binding affinity. By three ratios, Sld3CBD-Cdc45 demonstrated a significantly ssDNA residual level (t-test, ****p<0.0001) compared to other samples, indicating low binding affinity to ssDNA.
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Figure 4—source data 1
PDF file containing original native-PAGE for Figure 4, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig4-data1-v1.pdf
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Figure 4—source data 2
Original files for native-PAGE displayed in Figure 4.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig4-data2-v1.zip
Figure 4—figure supplement 1
Sequence of ssARS1 fragments.
(A) Fragments of ssARS1-1 through ssARS1-6 used in this study. Each ssARS1 fragment contains 80 bp. Fragments of odd and even numbers are complementary strands. The important elements A (autonomously replicating sequence, ARS consensus sequence), B1, B2, and B3 for unwinding are marked (Yuan et al., 2016). The fragments in the blue square are Sld3 binding parts of ssARS1. (B) The fragments of Sld3 binding parts (blue square parts in (A)) of ssARS1-2 and ssARS1-5. Each fragment of ssARS1-2 and ssARS1-5 is separated into 40 bp lengths: ssARS1-2-1 to ssARS1-2-3 and ssARS1-5-1 to ssARS1-5-3.
Figure 4—figure supplement 2
Electrophoresis mobility shift assay (EMSA) of ssDNA binding to Sld3 and its complexes with Sld7 and Cdc45.
(A) Single-stranded DNAs (ssDNAs) were visualized using Fast Blast DNA stain 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, 1:0.5, 1:1, 1:2, and 0:1, respectively. The controls for ssDNA and protein are lanes 1 and 5, respectively. The negative controls for no binding with ssDNA (ssARS 1–3_1, NC) of each sample are shown at the bottom. The reduce or disappearance of the ssDNA band in lanes 2–4 indicates that the protein (Sld3CBD, Sld7–Sld3ΔC, and Sld7–Sld3ΔC–Cdc45) binds to ssDNA with high affinity. The smeared bands appear in high molecular weight regions of lanes 2–4, when mixed with Sld7–Sld3ΔC–Cdc45 or Sld7–Sld3ΔC, whereas no bands appeared in the negative control (NC) (ssARS1-3_1). The positions of smeared ssDNA bonds correspond to those of protein in the protein-stain pages, indicating that ssARS1 was complexed with proteins. (B) The proteins were visualized using Coomassie brilliant blue on gels. The EMSA experiments were conducted concurrently under equivalent conditions to (A). The smeared bands in the high molecular weight parts of lanes 2–4 of Sld3CBD–Cdc45, Sld7–Sld3ΔC–Cdc45, and Sld7–Sld3ΔC are shown more clearly when mixed with ssDNA. Such enhanced discernibility indicates that these proteins easily enter the gel with ssDNA, even though Sld3CBD–Cdc45 binds ssDNA weakly. Sld3CBD could not enter the gel, even when bound to ssDNA, because the pI values exceeded the pH of the running buffer (pH = 8.3). Due to limitations in protein overexpression, we utilized Sld7–Sld3ΔC–Cdc45 and Sld7–Sld3ΔC from K. marxianus (same family as S. cerevisiae).
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Figure 4—figure supplement 2—source data 1
PDF file containing original native-PAGE for Figure 4—figure supplement 2, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig4-figsupp2-data1-v1.pdf
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Figure 4—figure supplement 2—source data 2
Original files for native-PAGE displayed in Figure 4—figure supplement 2.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig4-figsupp2-data2-v1.zip
Figure 4—figure supplement 3
DNA-binding assay by electrophoresis mobility shift assay.
(A) Different concentrations of Sld3CBD–Cdc45 and Sld7–Sld3ΔC–Cdc45 were incubated with ssDNA fragments. Lanes 1, 2, 3, and 4 represent ssDNA to protein ratios of 1:0, 0:1, 1:1, and 1:2, respectively. Lanes 1 and 2 are controls for single-stranded DNA (ssDNA) and protein, respectively. The DNAs were visualized using SYBR Safe on polyacrylamide gels. (B) Different concentrations of Sld7–Sld3ΔC–Cdc45 were incubated with the dsDNA fragment (dsARS1-34_1) mixed with jointed ssDNA fragments (ssARS1-2_1 or ssARS1-3_2). The ssARS1-2_1 and ssARS1-3_2 connect to dsARS1-34_1 at different sites. The control for ssDNA and double-stranded DNA (dsDNA) is located in lanes 1 and 2, respectively.
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Figure 4—figure supplement 3—source data 1
PDF file containing original native-PAGE for Figure 4—figure supplement 3, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig4-figsupp3-data1-v1.pdf
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Figure 4—figure supplement 3—source data 2
Original files for native-PAGE displayed in Figure 4—figure supplement 3.
- https://cdn.elifesciences.org/articles/101717/elife-101717-fig4-figsupp3-data2-v1.zip
Figure 4—figure supplement 4
Surface charge of Sld3CBD-Cdc45 and Sld3NTD-Sld7NTD.
The Sld3CBD-Cdc45 (A) and Sld3NTD-Sld7NTD (B) are presented in a charged surface calculated by the Pymol program. The blue and red show positive and negative charges, respectively. Cdc45 covers the main positive charge area of Sld3CBD α8CTP (A). A large positively charged region surrounds the middle of Sld7NTD in Sld3NTD-Sld7NTD (B).
Figure 4—figure supplement 5
Model of Sld3CTD on the Cdc45–MCM–GINS (CMG).
(A) Sld3CTD diagram on the SCM complex model. Sld3 could extend the C-terminal domain to interact with the Mcm4 NTD via the NTDs of Mcm2 and Mcm6. (B) Expansion of the Sld3CTD in close-up. The black dotted region denotes the extended region of Sld3CTD, which binds to the NTDs of Mcm2, Mcm4, and Mcm6. Sld3CBD C-terminal P420 is shown as red spheres. Labelled Mcm2, 4, and 6 subunits are colored cyan, blue, and purple blue, respectively. The subunit Mcm5 is colored dark blue, and the subunits Mcm3 and Mcm7 are colored gray. Green and pink are used to color Sld3CBD and Cdc45, respectively. GINS is shown in yellow, and a dsDNA is presented by the stick with dark orange.
Figure 5
Proposal for Cdc45–MCM–GINS (CMG) formation with Sld7–Sld3.
(A) Phosphorylation of Mcm2,4,6 by DDK after MCM double hexamer (DH) was loaded on double-stranded DNA (dsDNA) at the replication origin. (B) Cdc45 recruitment to MCM DH by Cdc45–Sld3–[Sld7]2–Sld3–Cdc45. (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 single-stranded DNA (ssDNA). (E) Each CMG unwinds the dsDNA in two directions, initiating DNA replication.
Additional files
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Supplementary file 1
Primers used in this study.
- https://cdn.elifesciences.org/articles/101717/elife-101717-supp1-v1.docx
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Supplementary file 2
Statistics of data collection and refinement.
- https://cdn.elifesciences.org/articles/101717/elife-101717-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/101717/elife-101717-mdarchecklist1-v1.docx
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Sld3CBD–Cdc45 structural insights into Cdc45 recruitment for CMG complex formation during DNA replication
eLife 13:RP101717.
https://doi.org/10.7554/eLife.101717.4
