Ordered and disordered regions of the Origin Recognition Complex direct differential in vivo binding at distinct motif sequences.

The Origin Recognition Complex (ORC) seeds replication-fork formation by binding to DNA replication origins, which in budding yeast contain a 17bp DNA motif. High resolution structure of the ORC-DNA complex revealed two base-interacting elements: a disordered basic patch (Orc1-BP4) and an insertion helix (Orc4-IH). To define the ORC elements guiding its DNA binding in vivo, we mapped genomic locations of 38 designed ORC mutants, revealing that different ORC elements guide binding at different sites. At silencing-associated sites lacking the motif, ORC binding and activity were fully explained by a BAH domain. Within replication origins, we reveal two dominating motif variants showing differential binding modes and symmetry: a non-repetitive motif whose binding requires Orc1-BP4 and Orc4-IH, and a repetitive one where another basic patch, Orc1-BP3, can replace Orc4-IH. Disordered basic patches are therefore key for ORC-motif binding in vivo, and we discuss how these conserved, minor-groove interacting elements can guide specific ORC-DNA recognition. Genome-wide binding of the wild-type and over 40 mutations of the Origin Replication Complex in budding yeast.

起始识别复合物（Origin Recognition Complex, ORC）通过结合DNA复制起点启动复制叉的形成，出芽酵母中的DNA复制起点包含一段17bp的DNA基序。ORC-DNA复合物的高分辨率结构揭示了两类碱基相互作用元件：一个无序碱性补丁（Orc1-BP4）与一个插入螺旋（Orc4-IH）。
为明确体内指导ORC与DNA结合的核心元件，我们对38种设计构建的ORC突变体的基因组结合位点进行了图谱分析，结果显示不同的ORC元件在不同位点介导结合过程。在缺失该基序的沉默相关位点中，ORC的结合与活性完全可通过BAH结构域进行解释。在复制起点内部，我们鉴定出两种占主导地位的基序变体，二者展现出差异化的结合模式与对称性：一类为非重复基序，其结合依赖Orc1-BP4与Orc4-IH的协同作用；另一类为重复基序，此时另一个碱性补丁Orc1-BP3可替代Orc4-IH发挥功能。
综上，无序碱性补丁是体内ORC识别结合基序的关键元件，我们还进一步讨论了这些保守的、能够与DNA小沟相互作用的元件如何介导特异性的ORC-DNA识别。本数据集包含出芽酵母中野生型起始识别复合物以及超过40种ORC突变体的全基因组结合数据。