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Conformational control and DNA-binding mechanism of the metazoan origin recognition complex [Biochemistry]
Proceedings of the National Academy of Sciences of the United States of America ( IF 11.1 ) Pub Date : 2018-06-26 , DOI: 10.1073/pnas.1806315115
Franziska Bleichert 1 , Alexander Leitner 2 , Ruedi Aebersold 2, 3 , Michael R Botchan 4 , James M Berger 5
Affiliation  

In eukaryotes, the heterohexameric origin recognition complex (ORC) coordinates replication onset by facilitating the recruitment and loading of the minichromosome maintenance 2–7 (Mcm2–7) replicative helicase onto DNA to license origins. Drosophila ORC can adopt an autoinhibited configuration that is predicted to prevent Mcm2–7 loading; how the complex is activated and whether other ORC homologs can assume this state are not known. Using chemical cross-linking and mass spectrometry, biochemical assays, and electron microscopy (EM), we show that the autoinhibited state of Drosophila ORC is populated in solution, and that human ORC can also adopt this form. ATP binding to ORC supports a transition from the autoinhibited state to an active configuration, enabling the nucleotide-dependent association of ORC with both DNA and Cdc6. An unstructured N-terminal region adjacent to the conserved ATPase domain of Orc1 is shown to be required for high-affinity ORC–DNA interactions, but not for activation. ORC optimally binds DNA duplexes longer than the predicted footprint of the ORC ATPases associated with a variety of cellular activities (AAA+) and winged-helix (WH) folds; cryo-EM analysis of Drosophila ORC bound to DNA and Cdc6 indicates that ORC contacts DNA outside of its central core region, bending the DNA away from its central DNA-binding channel. Our findings indicate that ORC autoinhibition may be common to metazoans and that ORC–Cdc6 remodels origin DNA before Mcm2–7 recruitment and loading.



中文翻译:

后生动物起源识别复合物的构象控制和DNA结合机制[生物化学]

在真核生物中,异六聚体起源识别复合物 (ORC) 通过促进微染色体维持 2-7 (Mcm2-7) 复制解旋酶的募集和加载到 DNA 上以许可起源来协调复制开始。果蝇ORC 可以采用一种自动抑制的配置,预计会阻止 Mcm2-7 的加载;复合物是如何被激活的以及其他 ORC 同源物是否可以呈现这种状态尚不清楚。使用化学交联和质谱、生化分析和电子显微镜 (EM),我们表明果蝇的自抑制状态ORC 填充在溶液中,人类 ORC 也可以采用这种形式。与 ORC 结合的 ATP 支持从自抑制状态到活性配置的转变,从而使 ORC 与 DNA 和 Cdc6 的核苷酸依赖性结合成为可能。与 Orc1 的保守 ATPase 结构域相邻的非结构化 N 末端区域被证明是高亲和力 ORC-DNA 相互作用所必需的,但不是激活所必需的。ORC 与 DNA 双链体的最佳结合时间比与多种细胞活动 (AAA + ) 和翼螺旋 (WH) 折叠相关的 ORC ATP 酶的预测足迹更长;果蝇的冷冻电镜分析ORC 与 DNA 和 Cdc6 结合表明 ORC 在其中心核心区域之外接触 DNA,使 DNA 弯曲远离其中心 DNA 结合通道。我们的研究结果表明,ORC 自身抑制可能对后生动物很常见,并且 ORC-Cdc6 在 Mcm2-7 募集和加载之前重塑了原始 DNA。

更新日期:2018-06-27
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