美国哈佛医学院Johannes C. Walter和英国爱丁堡大学Andrew P. Jackson团队合作取得一项新成果。他们利用计算机蛋白相互作用筛选揭示了DONSON在复制起始中的作用。相关论文于2023年8月17日发表在《科学》杂志上。

研究人员发现了DONSON，一种在小头原始侏儒症中突变的后生动物蛋白，是脊椎动物CMG组装所必需的。使用AlphaFold筛选蛋白质-蛋白质相互作用，然后进行实验验证，研究发现DONSON耦合脊椎动物前LC，其由GINS、TOPBP1和DNA pol ε构成。研究表明，DONSON将前LC对接到MCM2-7上，将GINS递送到CMG中的结合位点。患者来源的DONSON突变破坏了CMG组装，并重现了小鼠小头侏儒症的症状。

这些结果统一了人们对真核复制起始的理解，暗示了小头侏儒症中存在CMG组装缺陷，并证明计算机蛋白-蛋白质相互作用筛选可加速机制发现。

据介绍，CMG（Cdc45-MCM2-7-GINS）解旋酶组装是真核复制起始的中心事件。在酵母中，多亚基“预加载复合物”（pre-LC）伴随GINS与染色质结合的MCM2-7形成CMG。

附：英文原文

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

Author: Yang Lim, Lukas Tamayo-Orrego, Ernst Schmid, Zygimante Tarnauskaite, Olga V. Kochenova, Rhian Gruar, Sachiko Muramatsu, Luke Lynch, Aitana Verdu Schlie, Paula L. Carroll, Gheorghe Chistol, Martin A. M. Reijns, Masato T. Kanemaki, Andrew P. Jackson, Johannes C. Walter

Issue&Volume: 2023-08-17

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.

DOI: adi3448

Source: https://www.science.org/doi/full/10.1126/science.adi3448