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

• 成果类型: Article
• 署名作者: Lim, Yang; Tamayo-Orrego, Lukas; Schmid, Ernst; Tarnauskaite, Zygimante; Kochenova, Olga V.; Gruar, Rhian; Muramatsu, Sachiko; Lynch, Luke; Schlie, Aitana Verdu; Carroll, Paula L.; Chistol, Gheorghe; Reijns, Martin A. M.; Kanemaki, Masato T.; Jackson, Andrew P.; Walter, Johannes C.
• 署名单位: Harvard University; Harvard Medical School; University of Edinburgh; Howard Hughes Medical Institute; Research Organization of Information & Systems (ROIS); National Institute of Genetics (NIG) - Japan; Stanford University; Stanford University; Graduate University for Advanced Studies - Japan; University of Tokyo; UK Research & Innovation (UKRI); Biotechnology and Biological Sciences Research Council (BBSRC); Babraham Institute
• 刊物名称: SCIENCE
• ISSN/ISSBN: 0036-13290
• DOI: 10.1126/science.adi3448
• 发表日期: 2023-09-22
• 页码: 1301-+
• 关键词: chromosomal dna-replication; polymerase-alpha; complex; chromatin; association; cdc45; sld2; phosphorylation; checkpoint; inhibitor

摘要

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 epsilon. 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.