Cryo-EM structure of the human CTF18-RFC−PCNA complex in the presence of ATP.

a) Domain organization of the CTF18-RFC 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-RFC−PCNA complex, coloured by subunits. The less sharp definition of the map region corresponding to the AAA+ domain of the Ctf18 subunit supports a relative high mobility of this subunit. c) Molecular model of the CTF18-RFC−PCNA complex in ribbon representation. d) Structures of the human (left) and Saccharomyces cerevisiae (right) RFC−PCNA complex, which present the RFC pentamer in an auto-inhibited conformation analogous to the one captured in the CTF18-RFC−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. Residues that are not modelled in the cryo-EM structure are labelled as X in grey. 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 (45) are shown, highlighting the conservation of the β-hairpin fold. Below, the cryo-EM β-hairpin fold model is overlaid onto the corresponding motifs predicted by AlphaFold for Drosophila or Saccharomyces cerevisiae Ctf18. 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) subunit structures, with subdomains labelled. c) Structure of the Ctf18 subunit, in ribbon representation, within the CTF18-RFC−PCNA complex. The Ctf18 subdomains are coloured-coded as in panel (a). The other complex subunits are shown as surfaces.

Structural details of the Ctf18−RFC5 and CTF18-RFC−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 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-RFC−PCNA interactions, with CTF18-RFC subunit residues shown as sticks and PCNA as surface or ribbon. Polar interactions are shown as black dotted lines. The amino acid sequence encompassing the interacting motifs in the various subunits are shown.

Analysis of the auto-inhibited conformation of the CTF18 pentamer and nucleotide coordination.

a) Ribbon model of the CTF18-RFC pentamer (PCNA hidden). Nucleotide molecules are shown as sticks, with the corresponding cryo-EM density. b) Surface representation of (top) the CTF18-RFC pentamer and (bottom) the yeast RFC pentamer bound to open PCNA and primer-template DNA. PCNA is hidden in both structures. The overtwisted CTF18-RFC pentamer cannot accommodate DNA within the inner chamber. c) Model of the ATP active sites in the CTF18-RFC−PCNA complex, showing disruption of the interactions mediated by the arginine fingers. Below, insets show the nucleotide-binding site of subunit RFC4 in either the human CTF18-RFC (left) or human RFC (right) models, highlighting the structural incompatibility with nucleotide binding in the CTF18-RFC structure. Polar interactions with ATPγS in human RFC4 are indicated as cyan dotted lines. d) Detail of the inner chamber region of the CTF18-RFC 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. e) The structures of the RFC1 subunit from the yeast RFC–DNA–PCNA complex (11) and the Ctf18 subunit from the CTF18-RFC–PCNA complex are aligned to highlight conservation of the separation pin. Side chains of residues implicated in this motif in yeast RFC, along with the corresponding amino acids in CTF18-RFC, are shown.

Cryo-EM structure of the human CTF18-RFC−PCNA complex in the presence of ATP and Mg2+.

a) Two views of the Cryo-EM map, coloured 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.

Loading of PCNA onto P/T junctions by human clamp loader complexes.

a) Schematic representation of the FRET pair and experiment to monitor loading of Cy5-PCNA onto 5′ddPCy3/T DNA substrates engaged by RPA. The front and back faces of PCNA are displayed in green and grey, respectively. When loaded onto a 5′ddPCy3/T DNA substrate, the Cy5 label on the back face of PCNA is oriented towards the Cy3 label near the blunt duplex end of the 5′ddPCy3/T DNA substrate, yielding a FRET signal. b -d) Data. Each trace is the mean of at least three independent traces with the S.E.M. shown in grey. The time trajectories of I563 (magenta) and I665 (cyan) are displayed in the top panels and the corresponding EFRET (mustard) is displayed in the bottom panels. The time at which a clamp loader complex is added is indicated by a red arrow. Changes in I563 and I665 are indicated in the top panel by magenta and cyan arrows, respectively. The I563, I665, and the corresponding EFRET values observed prior to the addition of clamp loader complexes represents the complete absence of interactions between 5¢ddPCy3/T·RPA complexes and Cy5-PCNA. For observations, these values are each fit to flat line that is extrapolated to the axis limits. For observation, the I563 and I665 values observed after the addition of clamp loader complexes are fit to double exponential declines (I563) and rises (I665), respectively. The EFRET values observed after the addition of a given clamp loader are fit to double exponential rises and the respective rate constants (k1 and k2) are reported in the corresponding figure and in Table 1. Data for loading of PCNA by RFC, CTF18-RFC, and CTF18 Δ165-194 -RFC are displayed in panels b, c, and d, respectively.

Kinetic variables for loading of PCNA by human clamp loader complexes.

Kinetic variables are from the fits to double exponential rises for the data presented in Figure 6 in the main text. The values in parentheses indicate the relationship (as a fraction) between the rate constants observed for CTF18-RFC and CTF18 Δ165-194-RFC.

Primer extension assay with Pol ε and CTF18-RFC WT or CTF18△165-194-RFC.

a) Time course reactions on M13mp18 single-strand DNA with 40 nM wildtype CTF18-RFC or CTF18 Δ165-194-RFC. 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 Δ165-194-RFC. 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 in the Ctf18 large subunit slows down PCNA loading and reduces DNA synthesis by Pol ε.

a) The structure of the RFC module of CTF18-RFC is stabilized by the presence of a β-hairpin in the Ctf18 subunit (amino acids 165-194). Thus, wild type CTF18-RFC loads PCNA onto DNA efficiently and stimulates DNA synthesis by Pol ε interacting with the polymerase. b) The stability of the CTF18-RFC−PCNA complex is diminished by the deletion of the β-hairpin. Consequently, a CTF18-RFC deletion mutant lacking the β-hairpin (CTF18Δ165-194-RFC) shows slower loading of PCNA and lower activity in stimulating primer synthesis by Pol ε.