This study shows how the yeast Ctf4 protein couples the DNA helicase, Cdc45–MCM–GINS, to DNA polymerase α — the GINS subunit of the helicase and the polymerase use a similar interaction to bind Ctf4, suggesting that, as Ctf4 is a trimer, two polymerases could be simultaneously coupled to a single helicase during lagging-strand synthesis. During DNA replication, each polymerase is preceded by a helicase that disrupts the two strands, funnelling them into the leading- and lagging-strand machineries. This study, a collaboration between the laboratories of Luca Pellegrini, Alessandro Costa and Karim Labib, examines the structural basis for the action of the yeast Ctf4 protein that links DNA helicase and DNA polymerase components of the replisome. The authors delineate how Ctf4 couples the DNA helicase, Cdc45–MCM–GINS, to polymerase α. The GINS subunit of the helicase and the polymerase use a similar interaction to bind Ctf4, suggesting that, as Ctf4 is a trimer, two polymerases can be simultaneously coupled to a single helicase during lagging-strand synthesis. Efficient duplication of the genome requires the concerted action of helicase and DNA polymerases at replication forks1 to avoid stalling of the replication machinery and consequent genomic instability2,3,4. In eukaryotes, the physical coupling between helicase and DNA polymerases remains poorly understood. Here we define the molecular mechanism by which the yeast Ctf4 protein links the Cdc45–MCM–GINS (CMG) DNA helicase to DNA polymerase α (Pol α) within the replisome. We use X-ray crystallography and electron microscopy to show that Ctf4 self-associates in a constitutive disk-shaped trimer. Trimerization depends on a β-propeller domain in the carboxy-terminal half of the protein, which is fused to a helical extension that protrudes from one face of the trimeric disk. Critically, Pol α and the CMG helicase share a common mechanism of interaction with Ctf4. We show that the amino-terminal tails of the catalytic subunit of Pol α and the Sld5 subunit of GINS contain a conserved Ctf4-binding motif that docks onto the exposed helical extension of a Ctf4 protomer within the trimer. Accordingly, we demonstrate that one Ctf4 trimer can support binding of up to three partner proteins, including the simultaneous association with both Pol α and GINS. Our findings indicate that Ctf4 can couple two molecules of Pol α to one CMG helicase within the replisome, providing a new model for lagging-strand synthesis in eukaryotes that resembles the emerging model for the simpler replisome of Escherichia coli5,6,7,8. The ability of Ctf4 to act as a platform for multivalent interactions illustrates a mechanism for the concurrent recruitment of factors that act together at the fork.

DNA strand exchange, a process that helps to maintain the integrity of the genome, is promoted in eukaryotes by the Rad51 protein. Eukaryotes also have homologues of Rad51 with functions that are less well defined. Wolf-Dietrich Heyer and colleagues show that in yeast, two of these proteins, Rad55 and Rad57, form a complex that promotes recombination by stabilizing the Rad51 filament against the anti-recombinogenic activity of a helicase, Srs2. These results suggest that the Rad51 filament is a reversible intermediate, the stability of which reflects competing actions of other proteins. This mechanism may have relevance for the human RAD51 paralogues that have important roles in tumour suppression and disease. Homologous recombination is a high-fidelity DNA repair pathway. Besides a critical role in accurate chromosome segregation during meiosis, recombination functions in DNA repair and in the recovery of stalled or broken replication forks to ensure genomic stability. In contrast, inappropriate recombination contributes to genomic instability, leading to loss of heterozygosity, chromosome rearrangements and cell death. The RecA/UvsX/RadA/Rad51 family of proteins catalyses the signature reactions of recombination, homology search and DNA strand invasion1,2. Eukaryotes also possess Rad51 paralogues, whose exact role in recombination remains to be defined3. Here we show that the Saccharomyces cerevisiae Rad51 paralogues, the Rad55–Rad57 heterodimer, counteract the antirecombination activity of the Srs2 helicase. The Rad55–Rad57 heterodimer associates with the Rad51–single-stranded DNA filament, rendering it more stable than a nucleoprotein filament containing Rad51 alone. The Rad51–Rad55–Rad57 co-filament resists disruption by the Srs2 antirecombinase by blocking Srs2 translocation, involving a direct protein interaction between Rad55–Rad57 and Srs2. Our results demonstrate an unexpected role of the Rad51 paralogues in stabilizing the Rad51 filament against a biologically important antagonist, the Srs2 antirecombination helicase. The biological significance of this mechanism is indicated by a complete suppression of the ionizing radiation sensitivity of rad55 or rad57 mutants by concomitant deletion of SRS2, as expected for biological antagonists. We propose that the Rad51 presynaptic filament is a meta-stable reversible intermediate, whose assembly and disassembly is governed by the balance between Rad55–Rad57 and Srs2, providing a key regulatory mechanism controlling the initiation of homologous recombination. These data provide a paradigm for the potential function of the human RAD51 paralogues, which are known to be involved in cancer predisposition and human disease.

The trimeric spike (S) protein of SARS-CoV-2 is the primary focus of most vaccine design and development efforts. Due to intrinsic instability typical of class I fusion proteins, S tends to prematurely refold to the post-fusion conformation, compromising immunogenic properties and prefusion trimer yields. To support ongoing vaccine development efforts, we report the structure-based design of soluble S trimers with increased yields and stabilities, based on introduction of single point mutations and disulfide-bridges. We identify regions critical for stability: the heptad repeat region 1, the SD1 domain and position 614 in SD2. We combine a minimal selection of mostly interprotomeric mutations to create a stable S-closed variant with a 6.4-fold higher expression than the parental construct while no longer containing a heterologous trimerization domain. The cryo-EM structure reveals a correctly folded, predominantly closed pre-fusion conformation. Highly stable and well producing S protein and the increased understanding of S protein structure will support vaccine development and serological diagnostics.

The structure of the infectious prion protein (PrP^Sc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its insolubility and propensity to aggregate. PrP^Sc replicates by converting the non-infectious, cellular prion protein (PrP^C) into the misfolded, infectious conformer through an unknown mechanism. PrP^Sc and its N-terminally truncated variant, PrP 27–30, aggregate into amorphous aggregates, 2D crystals, and amyloid fibrils. The structure of these infectious conformers is essential to understanding prion replication and the development of structure-based therapeutic interventions. Here we used the repetitive organization inherent to GPI-anchorless PrP 27–30 amyloid fibrils to analyze their structure via electron cryomicroscopy. Fourier-transform analyses of averaged fibril segments indicate a repeating unit of 19.1 Å. 3D reconstructions of these fibrils revealed two distinct protofilaments, and, together with a molecular volume of 18,990 ų, predicted the height of each PrP 27–30 molecule as ~17.7 Å. Together, the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. Furthermore, they allow to formulate a molecular mechanism for the replication of prions. Knowledge of the prion structure will provide important insights into the self-propagation mechanisms of protein misfolding. The structure of the infectious prion (PrP^Sc), which is responsible for Creutzfeldt-Jakob disease in humans and bovine spongiform encephalopathy, has escaped all attempts at elucidation due to its propensity to aggregate. Here, we use the repetitive organization inherent in amyloid fibrils to analyze the structure of GPI-anchorless PrP 27–30 via electron cryomicroscopy. Fourier-transform analysis of averaged fibril segments indicates a repeating unit of 19.1 Å. In agreement with this observation, 3D reconstructions reveal that each fibril contains two distinct protofilaments and that the height of each PrP 27–30 molecule in these fibrils is ~17.7 Å. Together the data indicate a four-rung β-solenoid structure as a key feature for the architecture of infectious mammalian prions. The data conflict with all previous models for the structure of PrP^Sc and allow the formulation of a molecular mechanism for the replication of prions.

The Cdc45-MCM-GINS (CMG) helicase unwinds DNA during the elongation step of eukaryotic genome duplication and this process depends on the MCM ATPase function. Whether CMG translocation occurs on single- or double-stranded DNA and how ATP hydrolysis drives DNA unwinding remain open questions. Here we use cryo-electron microscopy to describe two subnanometre resolution structures of the CMG helicase trapped on a DNA fork. In the predominant state, the ring-shaped C-terminal ATPase of MCM is compact and contacts single-stranded DNA, via a set of pre-sensor 1 hairpins that spiral around the translocation substrate. In the second state, the ATPase module is relaxed and apparently substrate free, while DNA intimately contacts the downstream amino-terminal tier of the MCM motor ring. These results, supported by single-molecule FRET measurements, lead us to suggest a replication fork unwinding mechanism whereby the N-terminal and AAA+ tiers of the MCM work in concert to translocate on single-stranded DNA.