2. Academic Validation
  3. In silico protein interaction screening uncovers DONSON's role in replication initiation

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

  • Science. 2023 Aug 17;eadi3448. doi: 10.1126/science.adi3448.
Yang Lim # 1,  Lukas Tamayo-Orrego # 2,  Ernst Schmid 1,  Zygimante Tarnauskaite 2,  Olga V Kochenova 1 3,  Rhian Gruar 1,  Sachiko Muramatsu 4,  Luke Lynch 5,  Aitana Verdu Schlie 2,  Paula L Carroll 2,  Gheorghe Chistol 6,  Martin A M Reijns 2,  Masato T Kanemaki 4 7 8,  Andrew P Jackson 2,  Johannes C Walter 1 3
Affiliations

Affiliations

  • 1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Blavatnik Institute, Boston, MA 02115, USA.
  • 2 MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh, EH4 2XU, UK.
  • 3 Howard Hughes Medical Institute, Boston, MA 02115, USA.
  • 4 Department of Chromosome Science, National Institute of Genetics, Research Organization of Information and Systems (ROIS), Mishima, Shizuoka 411-8540, Japan.
  • 5 Biochemistry Department, Stanford School of Medicine, Stanford, CA 94305, USA.
  • 6 Chemical and Systems Biology Department, Stanford School of Medicine, Stanford, CA 94305, USA.
  • 7 Graduate Institute for Advanced Studies, SOKENDAI, Mishima, Shizuoka 411-8540, Japan.
  • 8 Department of Biological Science, The University of Tokyo, Tokyo 113-0033, Japan.
  • # Contributed equally.
PMID: 37590370 DOI: 10.1126/science.adi3448
Abstract

CMG (Cdc45-MCM2-7-GINS) 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.

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