Simon AC , Sannino V , Costanzo V , Pellegrini L .

614541 European Research Council, Wellcome Trust

	Figure 1. Crystal structure of human Cdc45.(a) The establishment of an active DNA replication fork requires Cdc45 association with MCM2-7 and formation of the CMG helicase assembly. (b) Top and side views of the Cdc45 structure, drawn as a ribbon with α-helices and β-strands in yellow and light blue, respectively. The putative path of the disordered region of human Cdc45 spanning residues 137–165 is drawn as grey dots. (c) Multiple sequence alignment of distantly related Cdc45 proteins. The secondary structure elements are shown as yellow and light blue boxes, numbered α1-20 for α-helices and β1-11 for β-strands and annotated as belonging to the DHH or DHHA1 domains, as appropriate. The four active site motifs of the DHH domain are included in boxes and marked I–IV. The extent of the disordered region preceding helix α6 in the DHH domain is indicated. Residues that were excised from the recombinant Cdc45 protein to promote crystallization are indicated by a dashed box. The position of F542 in the DHHA1 domain is marked by an asterisk.
	Figure 2. Human Cdc45 and bacterial RecJ.(a) Side-by-side comparison of the crystal structures of human Cdc45 (left) and bacterial RecJ (right; PDB ID 2ZXO), drawn as ribbons. The RecJ-homology fold is coloured yellow and orange in Cdc45 and RecJ, respectively, whereas the rest of the structure is coloured light grey. (b) Superposition of the RecJ-homology folds of Cdc45 and RecJ, coloured as in a. (c) Side-by-side comparison of Cdc45 and RecJ, highlighting the position of their DHH and DHHA1 domains, relative to the rest of their structures. The DHH and DHHA1 domains are coloured blue and red, respectively; helices 15 to 17, which connect the DHH and DHHA1 domains in human Cdc45 are coloured in light green.
	Figure 3. Intramolecular association of the DHH and DHHA1 domains.(a) Edge-on view of Cdc45, showing the position of the DHHA1 loop and the sidechain of F542, which mediates the intramolecular association of the DHHA1 and DHH domains in Cdc45. The DHH and DHHA1 domains are coloured light blue and red, respectively. Panels b and c show details of the hydrophobic and hydrophilic interactions at the DHH–DHHA1 interface, respectively. Hydrogen bonds are drawn in pink, and solvent molecules as small grey spheres. The distance of the polar contacts in panel c are shown in Ångström. (d) Side-by-side comparison of the RecJ (left; PDB ID 2ZXO) and Cdc45 (right). The DNA-binding groove of RecJ becomes inaccessible in Cdc45 because of the intramolecular association of its DHH and DHHA1 domains. The solvent-accessible surface of RecJ and Cdc45 is shown, coloured in light brown, whereas the DHH and DHHA1 domains are coloured blue and red, respectively.
	Figure 4. Docking of Cdc45 in the cryoEM map of the CMG.Panels a and b show top and bottom views of the Cdc45 structure fitted into the cryoEM map of fly CMG bound to DNA and ATPγS at 7.4 Å (EMDB ID 3318). Cdc45 is drawn as a ribbon, with the DHH and DHHA1 domains coloured blue and red, respectively, and the CMG map is shown as a grey, transparent envelope. The inset in each panel shows the CMG map, segmented and coloured to illustrate the position of each of its MCM2-7, GINS and Cdc45 subunits.
	Figure 5. The Cdc45–MCM interface.(a) The MCM-binding domain of Cdc45 is highlighted in red, relative to the rest of the Cdc45 structure in light grey. The structure is drawn as ribbons, with cylinders for α-helices. (b) The interface of Cdc45 with MCM2-7. The Cdc45 structure and homology models for the A-subdomain of MCM2 and MCM5 are shown as ribbons, docked in the map of fly CMG bound to DNA and ATPγS at 7.4 Å (EMDB ID 3318). The MCM-binding domain of Cdc45 is shown in red, the A-subdomains of MCM2 and MCM5 are shown in purple and pink, respectively, and the CMG map is drawn as a transparent envelope in light grey. The side chains of residues 319-GLPL-322 and F367, which are at the interface with MCM2 and MCM5, respectively, are also shown.
	Figure 6. The Cdc45–GINS interface.(a) The structural elements of the Cdc45 structure that come into contact with GINS in the CMG complex are highlighted in red, relative to the rest of the Cdc45 structure in light grey. The structure is drawn as ribbons with cylinders for α-helices. (b) View of the Cdc45–GINS interface that focuses on the interaction of the Psf1 B-domain with the DHH domain. Cdc45 is coloured as in a. The Psf1 subunit of GINS is shown in yellow, whereas the rest of the GINS structure (PDB ID 2Q9Q) is drawn in light grey. The Psf1 B-domain was modelled in Phyre2 and docked manually in the map of fly CMG bound to DNA and ATPγS at 7.4 Å (EMDB ID 3318); given the limited resolution of the map, its orientation relative to Cdc45 must be considered tentative. (c) View of the Cdc45–GINS interface that emphasises the contact between Psf2 and the acidic loop 256-NEDEENTLSVDC-267, linking α9 and β6 of the CID. Cdc45 is coloured as in a. The Psf2 subunit of GINS is shown in green, whereas the rest of the GINS structure is drawn in light grey.
	Figure 7. Structure-guided mutagenesis of Cdc45.The Cdc45 mutants chosen for functional analysis in Xenopus egg extracts are shown in the crystal structure of human Cdc45. The panels illustrate the position of the residues targeted for mutation, shown in blue on white space-fill model of the Cdc45 structure, docked in the cryoEM map of the of fly CMG bound to DNA and ATPγS at 7.4 Å (EMDB ID 3318), coloured in light brown. (a) Cdc45 mutants MCM1 to 3. (b) Cdc45 mutants GINS1 and GINS2. (c) Cdc45 mutant Helix6. (d) Cdc45 mutant Basic. (e) Cdc45 mutant targeting R406 (R407 in Xenopus Cdc45). The portion of the CMG map relative to the Psf1 B-domain was omitted for clarity.
	Figure 8. Structure-based functional characterization of Cdc45.(a) Wild-type (WT) and mutant Xenopus Cdc45 proteins were tested for their effect on DNA synthesis during DNA replication in Xenopus egg extracts. Each panel shows the results of the DNA synthesis assay, given as percentage over time of replicated DNA relative to the input (6,000 nuclei per μl). The assay for each mutant was performed in triplicate. The error bars represent the standard errors. The result of the assay for buffer addition is also shown. (b) Chromatin association of WT and mutant Xenopus Cdc45 proteins during DNA replication and its effect on the chromatin binding of other replication factors. Immunoblotting was carried out on isolated chromatin fractions after incubation for 30 min of sperm nuclei (4,000 nuclei per μl) in Xenopus interphase extracts supplemented either with buffer, WT or mutant Cdc45 protein at a final concentration of 100 ng μl−1. 1 μl of interphase extracts were loaded as input lane (Ext). Polyclonal rabbit anti-Xenopus Cdc45 was used to recognize the endogenous Cdc45 (xCdc45), as well as the recombinant WT and mutant proteins (rCdc45). Specific antibodies (described in the Methods) were used to evaluate the chromatin association level for Pol α, ORC1, MCM7, Psf3 and H2B.

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