Figures and data
Cryo-EM structure of the human CTF18-PCNA complex in the presence of ATP.
a) Domain organization of the CTF18 subunits forming the RFC pentamer. The dotted line above the Ctf18 subunit highlights the regions that are observed in the cryo-EM map. b) Two views of the cryo-EM map of the CTF18−PCNA complex, colored by subunits. The less sharp definition of the map region corresponding to the AAA+ domain of the Ctf18 subunit supports a relatively high mobility of this subunit. c) Molecular model of the CTF18−PCNA complex in ribbon representation. d) Structures of the human (left) (10) and Saccharomyces cerevisiae (right) RFC−PCNA complex (8), which present the RFC pentamer in an auto-inhibited conformation analogous to the one captured in the CTF18−PCNA complex.
Structure of the Ctf18 subunit.
a) Sequence of the human Ctf18 subunit, highlighting the secondary structural elements as assigned in the cryo-EM model. The atypical PCNA-interacting motif (PIP-box) and conserved DEXX box are boxed. Below the Ctf18 sequence to the left, the sequences of the N-terminus of human and other lower eukaryotes Ctf18, together with the secondary prediction by AlphaFold (42) are shown, highlighting the conservation of the β-hairpin fold. Below the Ctf18 sequence to the right, the alignment of the C-terminal region of human and Saccharomyces cerevisiae Ctf18, which interacts with the Ctf8/Dcc1 subunits, is shown. Asterisks denote conserved residues. b) Superposition of the human Ctf18 and RFC1 (PDB: 6VVO (10)) subunit structures, with subdomains labeled. c) Structure of the Ctf18 subunit, in ribbon representation, within the CTF18−PCNA complex. The Ctf18 subdomains are colored-coded as in panel (a). The other complex subunits are shown as surfaces
Structural details of the Ctf18-RFC5 and CTF18-PCNA interactions.
a) Model of the RFC5 collar domain and Ctf18 N-terminal β-hairpin in ribbon representation. The inset shows the interface in stick representation, with hydrogen bonds shown as green dotted lines. b) Map region around the Ctf18 N-terminal β-hairpin. c) Model of the PCNA homotrimer bound to the four CTF18 interacting regions. To the right, the structure of the yeast RFC−PCNA complex (11) with an open clamp, highlighting the engagement of all five RFC subunits to PCNA, required for clamp opening. d) Model of the regions of the CTF18−PCNA interactions, with CTF18 subunit residues shown as sticks and PCNA as ribbon.
Analysis of the auto-inhibited conformation of the CTF18 pentamer and nucleotide coordination.
a) Ribbon model of the CTF18 pentamer (PCNA hidden). Nucleotide molecules are shown as sticks, with the corresponding cryo-EM density. b) Surface representation of (top) the CTF18 pentamer and (bottom) the yeast RFC pentamer bound to open PCNA and primer-template DNA (11). PCNA is hidden in both structures. The overtwisted CTF18 pentamer cannot accommodate DNA within the inner chamber. c) Model of the ATP active sites in the CTF18−PCNA complex, showing disruption of the interactions mediated by the arginine fingers. d) Detail of the inner chamber region of the CTF18 pentamer, showing the β-hairpin (E-plug) of RFC5 protruding in the region predicted to be occupied by DNA. The DNA (grey ribbon) is modeled as in the yeast RFC−DNA−PCNA complex (11). e) The structures of the RFC1 subunit of the RFC−DNA−PCNA complex (11) and the Ctf18 subunit of the CTF18−PCNA complex are aligned to highlight the conservation of the separation pin interacting with DNA.
Cryo-EM structure of the human CTF18-PCNA complex in the presence of ATP and Mg2+.
a) Two views of the Cryo-EM map, colored by subunits. b) Molecular model in ribbon representation. The inset shows a map and model of the ATP molecule bound to the RFC2 subunit, with a discernible Mg2+ ion.
Primer extension assay with Pol ε and CTF18-RFC WT or CTF18-RFC△165-194.
a) Time course reactions on M13mp18 single-strand DNA with 40 nM CTF18-RFC or CTF18-RFCΔ165-194. Reactions were performed as described in Materials and Methods. b) Quantification of the band intensities from the primer extension assay shows that the rate of increase in intensity, derived from the initial linear portion of the reaction time before saturation, is 4142 ± 95 a.u./min for CTF18-RFC and 2389 ± 143 a.u./min for CTF18-RFCΔ165-194. This indicates that the mutant slows Pol ε synthesis by 42%. The experiment was conducted three times. Dots in b) represent mean values from the three replicas.
Deletion of the N-terminal β-hairpin of the CTF18 large subunit reduces DNA synthesis by Pol ε.
a) The structure of the RFC module of CTF18 is stabilized by the presence of a β-hairpin in the Ctf18 subunit (amino acids 165-194). Thus, wild type CTF18 stimulates DNA synthesis by Pol ε interacting with the polymerase and recruiting the sliding clamp PCNA. b) The stability of the CTF18−PCNA complex is compromised by the deletion of the β-hairpin. Consequently, a CTF18 deletion mutant lacking the β-hairpin (CTF18Δ165-194) shows lower activity in stimulating Pol ε primer synthesis.
SDS-PAGE gel (4-20%) of the purified human clamp loader CTF18 WT and CTF18Δ165-194.
Bands corresponding to the different subunits are labeled. The molecular weight standard is shown.
Cryo-EM of the CTF18−PCNA complex in the presence of ATP.
a) Representative electron micrograph acquired on a Falcon 4i electron detector in counting mode, and representative 2D class averages. b) Angular distributions of projections. c) Gold-standard Fourier shell correlation for the reconstruction of the full complex after postprocessing, and resolution estimation using the 0.143 criterion. d) Cryo-EM map of the complex, color-coded by local resolution.
Workflow of cryo-EM image processing and 3D reconstruction of CTF18-RFC-PCNA complex in the presence of 0.5 mM ATP and without Mg2+ (Dataset 1). Relion 4.0 was used for image processing and 3D reconstruction.
Walker A sequence and structure.
a) Walker A sequence comparison between CTF18 and hRFC (PDB: 6VVO). b) The upper panels illustrate the conformation of the Walker A motifs in Ctf18 and RFC1 (PDB: 6VVO). The lysine residue instead of valine does not seem to alter the loop conformation. The lower panels show the Walker A motif in the RFC3 subunit of CTF18 in the current study (left) and of hRFC (PDB: 6VVO) (right). Alterations in the motif appear to block ATP binding (10). S44 replaces the canonical proline, possibly preventing ATP binding.
Cryo-EM of the CTF18−PCNA complex in the presence of ATP and Mg2+.
a) Representative electron micrograph acquired on a Falcon 4i electron detector in counting mode, and representative 2D class averages. b) Angular distributions of projections. c) Gold-standard Fourier shell correlation for the reconstruction of the full complex after focused refinement, and resolution estimation using the 0.143 criterion. d) Cryo-EM map of the complex, color-coded by local resolution.
Workflow of cryo-EM image processing and 3D reconstruction of CTF18-RFC-PCNA complex in the presence of 0.5 mM ATP and 5 mM Mg2+ (Dataset 2). Relion 4.0 was used for image processing and 3D reconstruction.
Workflow of cryo-EM image processing and 3D reconstruction of CTF18-RFC△165-194-PCNA complex in the presence of 0.5 mM ATP and without Mg2+ (Dataset 3). Relion 5.0 was used for image processing and 3D reconstruction.
2D classification of CTF18 WT and CTF18-RFC△165-194.
a) The 2D classification of CTF18-RFC WT reveals the formation of the CTF18-RFC-PCNA complex with clear structural integrity (515 195 particles in all the showed 2D classes) b) The 2D classification of CTF18-RFC△165-194 mutant highlights the impact of β-hairpin deletion on complex stability. The presence of different orientations of the PCNA ring alone indicates that the absence of the β-hairpin contributes to the increased instability of the complex. Only the 2D classes squared in red represent the CTF18-RFC△165-194 -PCNA complex (14 616 particles).
Primer extension assays with Pol ε and CTF18 WT and CTF18-RFCΔ165-194.
CTF18-RFC or CTF18-RFCΔ165-194 concentration titration reactions on M13mp18 single-strand DNA. Reactions were running for 10 mins at 37°C.
