The interaction networks of the budding yeast and human DNA replication-initiation proteins

DNA replication is a stringently regulated cellular process. In proliferating cells, DNA replication-initiation proteins (RIPs) are sequentially loaded onto replication origins during the M-to-G₁ transition to form the pre-replicative complex (pre-RC), a process known as replication licensing. Subsequently, additional RIPs are recruited to form the pre-initiation complex (pre-IC). RIPs and their regulators ensure that chromosomal DNA is replicated exactly once per cell cycle. Origin recognition complex (ORC) binds to, and marks replication origins throughout the cell cycle and recruits other RIPs including Noc3p, Ipi1-3p, Cdt1p, Cdc6p and Mcm2-7p to form the pre-RC. The detailed mechanisms and regulation of the pre-RC and its exact architecture still remain unclear. In this study, pairwise protein-protein interactions among 23 budding yeast and 16 human RIPs were systematically and comprehensively examined by yeast two-hybrid analysis. This study tested 470 pairs of yeast and 196 pairs of human RIPs, from which 113 and 96 positive interactions, respectively, were identified. While many of these interactions were previously reported, some were novel, including various ORC and MCM subunit interactions, ORC self-interactions, and the interactions of IPI3 and NOC3 with several pre-RC and pre-IC proteins. Ten of the novel interactions were further confirmed by co-immunoprecipitation assays. Furthermore, we identified the conserved interaction networks between the yeast and human RIPs. This study provides a foundation and framework for further understanding the architectures, interactions and functions of the yeast and human pre-RC and pre-IC.

DNA复制是一个受到严格调控的细胞过程。在增殖细胞中，DNA复制起始蛋白（Replication Initiation Proteins, RIPs）会在M期至G₁期转变的过程中，依次加载至复制起点，进而形成前复制复合物（pre-replicative complex, pre-RC），这一过程被称为复制许可。后续，更多的RIPs会被招募以形成前起始复合物（pre-initiation complex, pre-IC）。RIPs及其调控因子确保染色体DNA在每个细胞周期中仅精确复制一次。起源识别复合物（Origin Recognition Complex, ORC）会结合并标记整个细胞周期中的复制起点，同时招募包括Noc3p、Ipi1-3p、Cdt1p、Cdc6p以及Mcm2-7p在内的其他RIPs，以组装形成pre-RC。目前，pre-RC的具体调控机制、组装细节以及其精确结构仍未明确。本研究通过酵母双杂交分析，系统且全面地检测了23种酿酒酵母RIPs与16种人类RIPs之间的成对蛋白质相互作用。本次研究共测试了470对酿酒酵母RIP相互作用组合以及196对人类RIP相互作用组合，分别鉴定得到113个和96个阳性相互作用。其中多数相互作用已被既往研究报道，但也存在部分全新的相互作用，包括各类ORC与MCM亚基间的相互作用、ORC的自身相互作用，以及IPI3和NOC3与多种pre-RC和pre-IC蛋白的相互作用。其中10种全新相互作用通过免疫共沉淀实验得到了进一步验证。此外，本研究还鉴定出了酿酒酵母与人类RIPs之间保守的相互作用网络。本研究为进一步理解酿酒酵母和人类pre-RC及pre-IC的结构、相互作用与功能提供了基础框架与研究依据。