Humanizing the Yeast Origin Recognition Complex

The evolutionarily conserved Origin Recognition Complex (ORC), plays a key role in origin selection in eukaryotes. However, ORC is strikingly divergent in its DNA binding specificity, ranging from base-specific interactions in Saccharomyces cerevisiae to base-agnostic interactions in humans. The mechanisms underlying this distinct selectivity is unknown. Atomic model of the yeast ORC showed that base-specific interaction with the invariant thymines of the ARS consensus sequence (ACS) is encoded by a 19-amino acid insertion helix (IH) embedded in the winged helix domain (WHD) of Orc4. This IH is absent in the Orc4 of metazoans including humans, suggesting that removal of the IH might give the yeast ORC “human-like” properties. Indeed, yeast strain engineered with IH deficient Orc4 has completely altered ORC-binding sites enriched in poly-dT tracts located in larger nucleosome-depleted and intergenic open chromatin. In vivo and in vitro assays show that the mutant ORC loads MCM efficiently, in spite of its altered specificity in favor of binding patterns more characteristic of those observed in humans/metazoans. This work provides insights for understanding how ORC evolves to adopt a life cycle that requires plasticity in origin selection during development.进化保守的起源识别复合物（Origin Recognition Complex, ORC）在真核生物的DNA复制起点选择过程中发挥核心作用。然而，ORC的DNA结合特异性存在显著分化：从酿酒酵母（Saccharomyces cerevisiae）的碱基特异性相互作用，到人类的碱基非依赖型相互作用，跨度极大。这种独特选择性背后的分子机制迄今仍未阐明。酵母ORC的原子结构模型显示，其与自主复制序列共识序列（ARS consensus sequence, ACS）中保守胸腺嘧啶的碱基特异性相互作用，由嵌入Orc4亚基翼螺旋结构域（winged helix domain, WHD）的19个氨基酸插入螺旋（insertion helix, IH）所编码。包括人类在内的后生动物的Orc4中并不存在该插入螺旋，这提示移除该插入螺旋或许能赋予酵母ORC“类人”的结合特性。经工程改造获得缺失插入螺旋的Orc4的酵母菌株，其ORC结合位点发生了全面重塑，转而富集于大型核小体缺失区域与基因间开放染色质内的多聚dT序列。体内与体外实验均证实，尽管该突变型ORC的结合特异性发生改变，转而偏向人类/后生动物中典型的结合模式，但其仍能高效装载微型染色体维持蛋白（Minichromosome Maintenance, MCM）。本研究为理解ORC如何演化以适应发育过程中需要复制起点可塑性的生命周期提供了重要见解。