A conserved mechanism for regulating replisome disassembly in eukaryotes

Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45–MCM–GINS (CMG) replicative helicase1–3. Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCFDia2 in budding yeast1, CUL2LRR1 in metazoa4–7), replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA8–10. However, it is unknown how SCFDia2 and CUL2LRR1 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome–E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR–MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity.

Replisome disassembly is the final step of eukaryotic DNA replication and is triggered by ubiquitylation of the CDC45-MCM-GINS (CMG) replicative helicase [1][2][3] . Despite being driven by evolutionarily diverse E3 ubiquitin ligases in different eukaryotes (SCF Dia2 in budding yeast 1 , CUL2 LRR1 in metazoa [4][5][6][7], replisome disassembly is governed by a common regulatory principle, in which ubiquitylation of CMG is suppressed before replication termination, to prevent replication fork collapse. Recent evidence suggests that this suppression is mediated by replication fork DNA [8][9][10] . However, it is unknown how SCF Dia2 and CUL2 LRR1 discriminate terminated from elongating replisomes, to selectively ubiquitylate CMG only after termination. Here we used cryo-electron microscopy to solve high-resolution structures of budding yeast and human replisome-E3 ligase assemblies. Our structures show that the leucine-rich repeat domains of Dia2 and LRR1 are structurally distinct, but bind to a common site on CMG, including the MCM3 and MCM5 zinc-finger domains. The LRR-MCM interaction is essential for replisome disassembly and, crucially, is occluded by the excluded DNA strand at replication forks, establishing the structural basis for the suppression of CMG ubiquitylation before termination. Our results elucidate a conserved mechanism for the regulation of replisome disassembly in eukaryotes, and reveal a previously unanticipated role for DNA in preserving replisome integrity. The eukaryotic replisome is assembled around the CMG helicase at replication origins during replication initiation. Once assembled, CMG remains stably associated with replication forks until two forks emanating from adjacent origins converge, or a single fork encounters the end of a linear chromosome or a template discontinuity, at which point replication terminates (Fig. 1a). Upon termination, the replisome is disassembled in two steps: first, CMG is ubiquitylated on its Mcm7 subunit by a cullin-RING E3 ubiquitin ligase (SCF Dia2 in budding yeast, CUL2 LRR1 in metazoa); second, ubiquitylated Mcm7 is unfolded by the Cdc48 ATPase (also known as p97 in higher eukaryotes), leading to disassembly of the replisome 1-3, [8][9][10] . As there is no known mechanism for origin-independent CMG assembly in S phase, premature disassembly of CMG must be avoided, to prevent replication fork collapse and genome instability 11 . CMG translocates on the leading-strand template while excluding the lagging-strand template from its central channel 12 . It has been suggested that this 'excluded' DNA strand, which is lost upon termination (Fig. 1a), inhibits ubiquitylation of CMG at replication forks [8][9][10] . However, because there are currently no structures of terminated replisomes in complex with SCF Dia2 or CUL2 LRR1 , how ubiquitylation of CMG is regulated to restrict replisome disassembly to termination remains a key unanswered question.

Terminated yeast replisome structures
To determine the molecular basis for the regulation of CMG ubiquitylation, we aimed to solve the structure of a terminated replisome, by adapting our system for reconstituting budding yeast replisomes for structural analysis 13 . After convergence of two replication forks, CMG translocates onto nascent double-stranded DNA (dsDNA) produced by the converging replisome 3,9,14 (Fig. 1a). To trap a replisome bound around dsDNA, we used a DNA substrate that lacked a 5′ flap and contained a short stretch methylphosphonate modifications embedded in dsDNA, which slow translocation of CMG 15 (Extended Data Fig. 1a). This DNA substrate was incubated with CMG, the replisome factors Tof1-Csm3, Mrc1 and Ctf4, SCF Dia2 (Hrt1-Cdc53-Skp1-Dia2), an E2-ubiquitin conjugate (Cdc34-Ub) 16 , and the leading-strand DNA polymerase Pol-ε, in the presence of ATP (Extended Data Fig. 1a). After glycerol gradient sedimentation, complexes containing all replisome and SCF Dia2 subunits were isolated (Extended Data Fig. 1b). Cdc34-Ub did not associate with the complex, perhaps reflecting the absence of neddylation on the Cdc53 cullin subunit of SCF Dia2 (refs. 17,18 ).
After gradient fixation, samples were prepared for cryo-electron microscopy (cryo-EM), yielding three-dimensional (3D) reconstructions at average resolutions of 3.2-4.0 Å (Fourier shell correlation (FSC) = 0.143 criterion; Extended Data Fig. 1c-h, Extended Data Table 1). DNA binding was heterogenous across the dataset, with the majority of particles still engaging single-stranded DNA (Extended Data Fig. 2). Nonetheless, we identified a subset of particles, which was subsequently subclassified into two conformations (conformations I and II), that had unambiguously translocated onto dsDNA, representative of bona fide termination intermediates produced after fork convergence Article (Fig. 1b, Extended Data Figs. 2, 3a-e). While the configuration of the MCM C-tier differed between conformations I and II (Extended Data Fig. 3), in both cases the incoming dsDNA was bent by approximately 46° between the MCM N-tier and C-tier, necessitating distortion of the DNA duplex within the N-tier (Extended Data Fig. 3f). For conformation I, the nucleotide occupancy and interactions with the phosphate backbone of the leading-strand template are similar to replication fork-bound CMG 13 (Fig. 1c, Extended Data Fig. 3b, g), suggesting a shared mechanism for translocation of CMG over single-stranded DNA and dsDNA 15 .
Having identified particles that had translocated onto dsDNA, we were able to build an atomic model of a terminated replisome (Fig. 1d). The overall architecture of CMG, Ctf4, Tof1-Csm3 and the non-catalytic module of Pol-ε (Pol-ε non-Cat ) was almost indistinguishable from previous structures 13, [19][20][21] (for details of the structure of Pol-ε, see Extended Data Fig. 4). We observed an additional, large region of density at the N-tier face of CMG beside Mcm3 and Mcm7, which closely approaches Csm3 and the dsDNA ahead of CMG, before extending away from the core of the complex, forming an elongated arm characteristic of the cullin subunit (Cdc53) of SCF Dia2 (Fig. 1d, e). The resolution of the cullin arm is relatively poor (precluding model building for Cdc53-Hrt1), due to a large degree of flexibility in this region, as highlighted by comparison of 3D classes (Extended Data Fig. 5a). We predict that this flexibility is important for conjugating the long K48-linked polyubiquitin chains required for Cdc48-dependent replisome disassembly 8 . Regardless, the orientation of SCF Dia2 can be unambiguously defined, placing the Cdc53 C terminus and Hrt1 ~45-70 Å from the primary ubiquitylation site on Mcm7 (Lys29) 8,22 (Extended Data Fig. 5a, b), consistent with previous structures of un-neddylated cullin-RING E3 ligases 23 .
Density corresponding to the E3 ligase substrate-recognition module (Skp1-Dia2) is adjacent to the N-tier face of CMG (Fig. 1d, e). The N-terminal tetratricopeptide repeat domain of Dia2, which binds Ctf4 and Mrc1 (refs. 8 When DNA replication terminates at the end of linear chromosomes, CMG is thought to dissociate from DNA, at which point the loss of the excluded strand triggers CMG ubiquitylation 8,9 (Fig. 1a). To establish how SCF Dia2 engages the replisome following termination at chromosome ends, we repeated cryo-EM sample preparation as described above, except in the absence of DNA. This yielded a 3D reconstruction of an 'off DNA' replisome at 3.9 Å resolution (Fig. 1f, Extended Data Fig. 6). Notably, binding of the Dia2 LRRs across Mcm3, Mcm5 and Mcm7 is indistinguishable from complexes bound around dsDNA (Fig. 1g, Extended Data Fig. 3e). Furthermore, comparison of our dsDNA-bound and off-DNA complexes with a previous structure of a replication fork-associated replisome 13 revealed no conformational changes in the region of the MCM N-tier to which Dia2 binds (Fig. 1g, Extended Data Fig. 3e). Therefore, we conclude that termination does not induce conformational changes in CMG that are important for the regulation of CMG ubiquitylation by SCF Dia2 (ref. 26 ), either following fork convergence or when CMG dissociates from DNA.

Dia2 LRR -MCM interface
The extensive interface between Dia2 LRR and MCM is predominantly formed by the Mcm3 N-tier (helices α1 and α5 and the zinc-finger (ZnF)    (Fig. 2d). Together, these data demonstrate that the Dia2 LRR -MCM interface that we describe is essential for CMG ubiquitylation and replisome disassembly, both after fork convergence and when CMG dissociates from DNA.
The overall architecture of human CMG, AND-1, TIMELESS-TIPIN and Pol-ε are indistinguishable from our previous structure lacking CUL2 LRR1 (ref. 28 ) (Fig. 3a, b, Extended Data Fig. 10a). LRR1 is positioned across the MCM N-tier, in close proximity to the parental DNA duplex. In addition, an elongated arm of lower-resolution density, into which the IPs of Sld5 (and CMG) Article crystal structure of ELOB-ELOC-CUL2-RBX1 could be unambiguously docked 29 , projects from the MCM N-tier in an analogous manner to yeast Cdc53-Hrt1 (Fig. 3b). Although metazoan CUL2 and yeast Cdc53 are tethered to their respective substrate adaptors (ELOB-ELOC-LRR1 for CUL2, Skp1-Dia2 for Cdc53) via very different interactions, the cullin C terminus and RING-box protein are similarly located in both cases, ~45-70 Å from the primary ubiquitylation sites in Mcm7 (refs. 8,9,22 ) (Figs. 1d, 3b). Furthermore, like Cdc53, CUL2 displays considerable conformational variability, which is probably important for the conjugation of long polyubiquitin chains onto MCM7 (refs. 8,30 ) (Extended Data Figs. 5a, 10b). The majority of LRR1 was well resolved in our cryo-EM map (Extended Data Fig. 9e, k-n), which enabled de novo modelling of an N-terminal pleckstrin homology domain and a C-terminal LRR domain, which are connected by a flexible linker that stretches perpendicularly across the parental dsDNA (Fig. 3c, d). The pleckstrin homology domain interacts with the ZnF domains of MCM2 and MCM6, parental dsDNA and the N-terminal region of the TIMELESS α-solenoid (Extended Data Fig. 10c,  d), consistent with the reported role for TIMELESS-TIPIN in recruiting CUL2 LRR1 to the replisome in Caenorhabditis elegans 30 . The LRR domain comprises seven canonical and two irregular LRR motifs and forms a shallow arc, reaching from the parental dsDNA to the N-tier face of MCM3 and MCM5 (Fig. 3d, Extended Data Fig. 10e, f). The BC and CUL2 boxes, which link LRR1 to ELOB-ELOC-CUL2-RBX1, are situated between LRR repeats 8 and 9 (Fig. 3c, d, Extended Data Fig. 10e, f), and a two-stranded antiparallel β-sheet caps the LRR domain at its C-terminal end (Extended Data Fig. 10g). In addition, the C-terminal HMG box of AND-1 could be docked into a small region of density alongside ELOC and LRR1 (Extended data Fig. 10h, i), which was absent in 3D classes that lacked AND-1 (Extended Data Fig. 10j, k), indicating that AND-1 interacts with CUL2 LRR1 in the human replisome.
Remarkably, despite the very different architectures of the LRR1 and Dia2 LRR domains, they bind to the same region of the MCM N-tier, but do so via completely different modes of interaction. The LRR1 LRR domain interacts predominantly with the three-stranded antiparallel β-sheet of the ZnF domain of MCM3, which extends the shallow arc of the LRR1 β-sheet (Extended Data Fig. 10l). This interface is augmented on one side by interactions between the MCM7 N terminus and the tip of the ZnF domain of MCM3 and LRR1 repeats 8 and 9 (Extended Data Fig. 10l, m). On the other side, MCM3 residues 3-8 and 164-174 are significantly rearranged upon CUL2 LRR1 binding, such that the N terminus of MCM3, now projecting between the ZnF domains of MCM3 and MCM5, stabilizes an interaction between MCM3 residues 164-174 and a loop and short helix preceding LRR1 repeat 9 (Extended Data Fig. 10n). Finally, charged residues immediately preceding the β-strands of LRR1 repeats 4-7 form multiple polar contacts with the tip of the ZnF domain of MCM5 (Extended Data Fig. 10m). Further details are illustrated in Extended Data Fig. 10m, n.

Regulation of CMG ubiquitylation
Ubiquitylation of CMG by both SCF Dia2 and CUL2 LRR1 is suppressed by the excluded DNA strand at replication forks 8-10 ; our discovery that Dia2 and LRR1 bind directly to a common site across the ZnF domains of MCM3 and MCM5 suggested that this region of MCM might be important for the regulation of ubiquitylation. In our recent structure of the human replisome bound to a replication fork 28 , cryo-EM density that we attributed to the excluded strand was positioned in the channel between the ZnF domains of MCM3 and MCM5, consistent with previous structures of Drosophila and budding yeast CMG 13,31,32 . To further validate our assignment of the excluded strand, we identified a subset of particles lacking CUL2 LRR1 from our dataset of replisomes assembled without an excluded strand (Extended Data Figs. 8, 9g). In the resulting density map, the MCM N-tier was identical to our previous map of replication fork-associated CMG 28 , apart from a single region of density, extending from the fork junction between the ZnF domains of MCM3 and MCM5 (Fig. 4a, Extended Data Fig. 10o). This density was present only in the complex associated with the replication fork, thus confirming that it is contributed by the excluded DNA strand.
Crucially, Fig. 4b shows that the presence of the excluded strand between the ZnF domains of MCM3 and MCM5 sterically blocks the engagement of the Dia2 and LRR1 LRR domains with MCM. As the LRR-MCM interaction is essential for ubiquitylation of CMG and, in turn, replisome disassembly, the occlusion of this interface by the excluded strand provides an elegant and universal explanation for the regulation of replisome disassembly across yeasts and metazoa. Notably, the LRR domains of Dia2 and LRR1 are not demonstrably homologous in sequence or structure. Thus, we propose that the binding of Dia2 LRR and LRR1 LRR across the exit channel of the excluded strand reflects convergent evolution, probably indicative of a stringent evolutionary pressure to accurately regulate replisome disassembly, and thereby safeguard replication forks. This evolutionary constraint is not evident in parts of the replisome disassembly machinery that do not contribute to the regulation of CMG ubiquitylation. For example, the Dia2 tetratricopeptide repeat domain binds yeast Mrc1 and Ctf4, whereas the LRR1 pleckstrin homology domain binds human TIMELESS.
On the basis of our results, we propose the model summarized in Fig. 4c. Ubiquitylation of the MCM double hexamer is blocked by the occlusion of the LRR binding site at the inter-hexamer interface 27 (Extended Data Fig. 11a). This occlusion probably also suppresses ubiquitylation during the conversion of MCM double hexamers into pairs of active CMG helicases 33 , before the lagging-strand template is excluded. Once bidirectional replication forks are established and elongation begins, the spooling of the excluded DNA strand between the ZnF domains of MCM3 and MCM5 sterically blocks LRR engagement on MCM. It is possible that the binding of proteins to the excluded strand may help to block LRR-MCM engagement. However, ubiquitylation of CMG is inhibited at reconstituted budding yeast replication forks in the absence of the lagging-strand machinery (Extended Data Fig. 11b, c), consistent with the excluded DNA alone being sufficient to suppress SCF Dia2 during elongation. In principle, the binding of yeast Dia2 to Mrc1 and Ctf4, and human LRR1 to TIMELESS and AND-1, could still occur at replication forks, even when the LRR-MCM interaction is blocked by the excluded strand. Accordingly, Mrc1-Ctf4 can support SCF Dia2-13A recruitment to reconstituted replisomes (Extended Data Fig. 11d, e). Critically, however, the essentiality of the LRR-MCM interaction for ubiquitylation of CMG will restrict replisome disassembly to termination, independent of the timing of E3 ligase recruitment, and irrespective of whether a replication fork terminates via fork convergence, or at a telomere.
Finally, we note that if the excluded strand is ever mispositioned, for example, during replication fork stalling or reversal, replisome disassembly could be triggered, due to premature LRR-MCM engagement. As such, the regulatory mechanism that we describe here may have implications for the stability of the replication fork under conditions of replication stress.

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Cryo-EM density maps of the yeast replisome-SCF Dia2   A description of all covariates tested A description of any assumptions or corrections, such as tests of normality and adjustment for multiple comparisons A full description of the statistical parameters including central tendency (e.g. means) or other basic estimates (e.g. regression coefficient) AND variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals) For null hypothesis testing, the test statistic (e.g. F, t, r) with confidence intervals, effect sizes, degrees of freedom and P value noted For manuscripts utilizing custom algorithms or software that are central to the research but not yet described in published literature, software must be made available to editors and reviewers. We strongly encourage code deposition in a community repository (e.g. GitHub). See the Nature Portfolio guidelines for submitting code & software for further information.

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Policy information about availability of data All manuscripts must include a data availability statement. This statement should provide the following information, where applicable: -Accession codes, unique identifiers, or web links for publicly available datasets -A description of any restrictions on data availability -For clinical datasets or third party data, please ensure that the statement adheres to our policy March 2021 EMD-13539 (conformation II). Cryo-EM density maps of the yeast replisome-SCFDia2 complex in the absence of DNA have been deposited in the EMDB under the following accession numbers: EMD-13519 (full complex unsharpened map) and EMD-13540 (MBR). Cryo-EM density maps of the human replisome-CUL2LRR1 complex used in model building have been deposited in the EMDB under the following accession numbers: EMD-13494 (full complex, consensus refinement), EMD-13491 (MBR, AND-1/CDC45/GINS), EMD-13490 (MBR, ELONGIN-BC/LRR1/CUL2), EMD-13492 (MBR, CUL2-RBX1). An additional map of the core human replisome not engaged by CUL2LRR1 on a DNA substrate lacking a 5'-flap has been deposited under the accession number EMD-13534. Atomic coordinates have been deposited in the Protein Data Bank (PDB) with the accession numbers 7PMK for the yeast replisome-SCFDia2 complex on dsDNA (conformation I), 7PMN for the yeast replisome-SCFDia2 complex on dsDNA (conformation II), and 7PLO for the human replisome-CUL2LRR1 complex.

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Sample size
Our study does not include cohort/population based analysis or comparison and thus does not entail predetermination of sample size. Cryo-EM data were collected, as described in methods; these numbers of micrographs were sufficient to either allow model building or comparative analysis.
Data exclusions During processing of cryo-EM data, poor quality micrographs/particles were excluded based on manual inspection and 2D/3D classification.

Replication
Cryo-EM datasets for yeast and human complexes comprised individual sample preparations and datasets. Complex formation was found to be reproducible across multiple independent sample preparations. Details of the number of experimental repeats have been acknowledged in the relevant figure legends. All attempts at data replication were successful. The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
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Methodology
Sample preparation For each sample, 10 million yeast cells were harvested and fixed by resuspension in 1 ml of 70% ethanol. Subsequently, 3 ml of 50 mM sodium acetate and 50mg of RNase A was added to 150 uL of fixed cells, followed by incubation at 37°C for 2 h. The cells were then pelleted and proteins degraded by incubation at 37°C for 30 min in 500 uL of 50 mM HCl containing 2.5 mg of Pepsin. Finally, cells were pelleted and then re-suspended in 1 ml of 50 mM sodium citrate containing 2 mg of propidium iodide. Samples were sonicated and then analysed.

Cell population abundance
The percentage of cells in G1-phase is provided in EDF 7i.

Gating strategy
Gating was performed to remove cell debris and dead cells based on forwards and side scatter properties, as exemplified in Supplementary Fig. 2 Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.