Comprehensive Characterization of Minichromosome Maintenance Complex (MCM) Protein Interactions Using Affinity and Proximity Purifications Coupled to Mass Spectrometry

The
extensive identification of protein–protein interactions
under different conditions is an important challenge to understand
the cellular functions of proteins. Here we use and compare different
approaches including affinity purification and purification by proximity
coupled to mass spectrometry to identify protein complexes. We explore
the complete interactome of the minichromosome maintenance (MCM) complex
by using both approaches for all of the different MCM proteins. Overall,
our analysis identified unique and shared interaction partners and
proteins enriched for distinct biological processes including DNA
replication, DNA repair, and cell cycle regulation. Furthermore, we
mapped the changes in protein interactions of the MCM complex in response
to DNA damage, identifying a new role for this complex in DNA repair.
In summary, we demonstrate the complementarity of these approaches
for the characterization of protein interactions within the MCM complex.

在不同生理条件下大规模鉴定蛋白质-蛋白质相互作用，是解析蛋白质细胞功能的重要研究挑战。本研究采用并对比了包括亲和纯化（affinity purification）、邻近偶联质谱纯化在内的多种方法，用于鉴定蛋白质复合物。我们针对所有不同的微染色体维持复合物（minichromosome maintenance, MCM）蛋白，同时采用上述两种方法，解析了该复合物的完整相互作用组（interactome）。总体而言，本研究的分析结果不仅鉴定出了特异性与共有的相互作用伴侣蛋白，还发现这些蛋白显著富集于DNA复制、DNA修复及细胞周期调控等不同生物学过程。此外，我们还绘制了DNA损伤应答条件下MCM复合物蛋白质相互作用的动态变化，并揭示了该复合物在DNA修复中此前未被报道的新功能。综上，本研究证实了这两种方法在表征MCM复合物内部蛋白质相互作用时具有良好的互补性。