XB-ART-60319
Science
2023 Sep 22;3816664:eadi3448. doi: 10.1126/science.adi3448.
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In silico protein interaction screening uncovers DONSON's role in replication initiation.
Lim Y
,
Tamayo-Orrego L
,
Schmid E
,
Tarnauskaite Z
,
Kochenova OV
,
Gruar R
,
Muramatsu S
,
Lynch L
,
Schlie AV
,
Carroll PL
,
Chistol G
,
Reijns MAM
,
Kanemaki MT
,
Jackson AP
,
Walter JC
.
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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.
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Howard Hughes Medical Institute , R01 HL098316 NHLBI NIH HHS , R35 GM147060 NIGMS NIH HHS , T32 GM007276 NIGMS NIH HHS , MC_UU_00035/10 Medical Research Council
Genes referenced: cdc45 mcm2 topbp1
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