In:
Science, American Association for the Advancement of Science (AAAS), Vol. 381, No. 6664 ( 2023-09-22)
Abstract:
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.
Type of Medium:
Online Resource
ISSN:
0036-8075
,
1095-9203
DOI:
10.1126/science.adi3448
Language:
English
Publisher:
American Association for the Advancement of Science (AAAS)
Publication Date:
2023
detail.hit.zdb_id:
128410-1
detail.hit.zdb_id:
2066996-3
detail.hit.zdb_id:
2060783-0
SSG:
11
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