In silico protein interaction screening uncovers DONSON’s role in replication initiation

CDC45-MCM2-7-GINS (CMG) helicase assembly is the central event in eukaryotic replication initiation. In yeast, a multi-subunit “pre-loading complex” (pre-LC) accompanies GINS to chromatin-bound MCM2-7, leading to CMG formation. Here, we report that DONSON, a metazoan protein mutated in microcephalic primordial dwarfism, is required for CMG assembly in vertebrates. Using AlphaFold to screen for protein-protein interactions followed by experimental validation, we show that DONSON scaffolds a vertebrate pre-LC containing GINS, TOPBP1, and DNA pol ε. Our evidence suggests that DONSON docks the pre-LC onto MCM2-7, delivering GINS to its binding site in CMG. A patient-derived DONSON mutation compromises CMG assembly and recapitulates microcephalic dwarfism in mice. These results unify our understanding of eukaryotic replication initiation, implicate defective CMG assembly in microcephalic dwarfism, and illustrate how in silico protein-protein interaction screening accelerates mechanistic discovery. Description Editor’s summary Chromosomes are copied only once during each cell division to allow inheritance of the genetic blueprint by the next generation. Until now, our understanding of how cells assemble the molecular machinery for chromosome duplication has relied heavily on studies of yeast. AlphaFold-Multimer (AF-M) is an artificial intelligence-based system that can predict whether proteins interact. Lim et al. applied this technology to address the function of an enigmatic protein called DONSON, which was previously implicated in DNA replication. They used AF-M to predict which of the 20,000 proteins expressed in human cells bind DONSON. Combined with experimental validation, this in silico screen indicated that DONSON is the missing link in the assembly of the complex that unzips the DNA double helix during replication. Working in the roundworm Caenorhabditis elegans, Xia et al. showed that the worm homolog of DONSON, DNSN-1, is essential for the assembly of the replication machinery. Homologs of DNSN-1 are absent from yeast but present in plants and animals. Mutations of the equivalent protein in humans causes a form of microcephalic dwarfism often seen when assembly of the chromosome duplication machinery is defective. —Di Jiang AlphaFold predictions implicate the protein DONSON as a key player in vertebrate replication initiation. INTRODUCTION Rapid and faithful DNA replication maintains genome integrity and is often disrupted in human diseases such as cancer. A key event during replication initiation is the assembly of the CDC45-MCM2-7-GINS (CMG) helicase, which unwinds DNA at the fork and is composed of the hexameric MCM2-7 ATPase and two accessory factors, CDC45 and GINS. After MCM2-7 double hexamers are recruited to origins in G1 and cells enter S phase, CDC45 and GINS associate sequentially with MCM2-7 double hexamers, leading to CMG assembly. In yeast, GINS is escorted to MCM double hexamers by a “pre-loading complex” (pre-LC) containing Dpb11, Sld2, and DNA polymerase ε (Pol ε), but how GINS is recruited to origins in metazoa remains unclear. In addition, multicellular organisms contain proteins such as Downstream Neighbor of SON (DONSON) that impact replication through unknown mechanisms. RATIONALE We wanted to understand how DONSON, which is mutated in microcephalic dwarfism, contributes to DNA replication. To this end, we used a combination of biochemistry, cell biology, mouse genetics, and in silico screening for novel protein-protein interactions. RESULTS We first immunodepleted DONSON from cell-free Xenopus laevis egg extracts, which recapitulate genome maintenance processes. We found that in the absence of DONSON, DNA replication and CMG assembly were abolished. Using an inducible DONSON degron allele, we showed that DONSON is also essential for CMG assembly and DNA replication in human cells. To explore how DONSON promotes CMG formation, we searched the entire replisome for DONSON-interacting proteins using AlphaFold-Multimer (AF-M). This focused in silico screen identified GINS, TOPBP1 (Dpb11 ortholog), POLE2 (Pol ε subunit), MCM3 (MCM2-7 subunit), and DONSON itself as high confidence DONSON interactors. Based on these predictions, we postulated that DONSON scaffolds a dimeric vertebrate pre-LC containing two copies of DONSON, TOPBP1, GINS, and Pol ε, and that this pre-LC positions GINS on CDC45-MCM2-7 for CMG assembly (see figure). Consistent with this model, biochemical studies confirmed that DONSON’s interactions with TOPBP1 and GINS are essential for CMG formation and DNA replication. On the other hand, DONSON’s interaction with MCM3 was dispensable for pre-LC assembly and instead promoted pre-LC docking onto MCM double hexamers. A DONSON mutation that causes microcephalic dwarfism in humans recapitulated this condition in mice and compromised both replication initiation and CMG assembly. Finally, when we screened the entire human proteome for DONSON interactors using AF-M and additional criteria, CMG assembly factors were highly enriched, suggesting the potential of in silico screening for ab initio protein function identification. CONCLUSION We identify DONSON as a factor required for vertebrate CMG assembly. It functions by organizing a pre-LC that delivers GINS to its binding site on CDC45-MCM2-7, leading to CMG assembly. Our results suggest that although all eukaryotes contain a pre-LC, their architectures differ, with vertebrates using DONSON instead of Sld2. Our results also implicate defective CMG assembly in the pathophysiology of microcephalic dwarfism. We propose that impaired replication initiation contributes to slow cell cycle progression, leading to the hypocellularity seen in this form of dwarfism. Our results illustrate that large-scale in silico protein-protein interaction screening using AF-M is an effective method to identify functionally relevant interactions and formulate testable molecular hypotheses. DONSON scaffolds a pre-Loading complex that delivers GINS to CDC45-MCM2-7 for CMG assembly. (A) Steps taken to generate and validate a model of how DONSON promotes CMG assembly. (B) An AlphaFold model of the dimeric pre-LC is shown docking onto the CDC45-MCM2-7 double hexamer, leading to formation of two CMGE (CMG + Pol ε) complexes. We propose that pre-LC docking requires separation of the two MCMs and rotation of one MCM relative to the other. Whether DONSON remains associated with CMGE, as shown, is unclear. Several subunits of Pol ε and most domains of TOPBP1 are not shown nor are flexible loops connecting the different parts of DONSON and TOPBP1.