Conformational control and DNA-binding mechanism of the metazoan origin recognition complex
Conformational control and DNA-binding mechanism of the metazoan origin recognition complex
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DOI：
10.1073/pnas.1806315115
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发表时间：
2018-06-26
影响因子：
11.1
通讯作者：
Berger, James M.
中科院分区：
文献类型：
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作者：
Bleichert, Franziska;Leitner, Alexander;Berger, James M.
在真核生物中，异六聚体起源识别复合体（ORC）通过促进小染色体维持2-7 （Mcm2-7）复制解旋酶在DNA上的招募和装载来协调复制的开始，以许可起源。果蝇ORC可以采用自抑制结构，预计可以阻止Mcm2-7加载；复合物是如何被激活的，以及其他ORC同源物是否可以处于这种状态尚不清楚。通过化学交联、质谱分析、生化分析和电子显微镜（EM），我们发现果蝇ORC在溶液中存在自抑制状态，人类ORC也可以采用这种形式。ATP结合到ORC支持从自抑制状态到活性配置的转变，使ORC与DNA和Cdc6的核苷酸依赖关联成为可能。Orc1保守的atp酶结构域附近的非结构化n端区域被证明是高亲和ORC-DNA相互作用所必需的，但不是激活所必需的。ORC结合DNA双链的最佳长度比ORC atp酶与各种细胞活动（AAA(+)）和翼状螺旋（WH）折叠相关的预测足迹长；对与DNA和Cdc6结合的果蝇ORC的冷冻电镜分析表明，ORC与其核心区域外的DNA接触，使DNA弯曲，远离其中心DNA结合通道。我们的研究结果表明，ORC自身抑制可能在后生动物中很常见，并且ORC- cdc6在Mcm2-7招募和装载之前重塑了起源DNA。
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.