Filament Formation Induces a Shape Change and Activation of the Nuclease SgrAI
Filament Formation Induces a Shape Change and Activation of the Nuclease SgrAI
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丝状形成引起形状变化和核酸酶 SgrAI 的激活
DOI：
10.1096/fasebj.2020.34.s1.00730
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Horton, Nancy C.
中科院分区：
文献类型：
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作者：
Horton, Nancy C.
SgrAI是变构的，当结合到DNA中的主要识别序列时，SgrAI既被激活，又经历底物特异性的变化(切割第二类DNA序列)。我们用结构、生物物理和酶动力学方法研究了SgrAI的变构激活和这种DNA序列特异性的异常扩展。1-2各种生物物理技术(天然凝胶电泳法、分析超速离心法、离子迁移率天然质谱仪、X射线结晶学和电子显微镜)以研究SgrAI形成的复合体。用单周转DNA裂解实验研究了SgrAI的反应机理，并通过全局建模建立了包括每个重要步骤的速率常数的全反应路径计算模型。生物物理表征表明，SgrAI在激活时会形成Run-On寡聚体或细丝，当SgrAI与其主要识别序列结合时，这些丝会被诱导出来。SgrAI与二级位点序列紧密结合，二级位点序列与初级位点序列相差单个碱基对，但无法切割它们，也不能诱导微丝形成。然而，结合到次级位点序列的SgrAI将连接由结合到初级位点序列的SgrAI形成的细丝。SgrAI/DNA复合体之间的蛋白质-蛋白质和蛋白质-DNA接触稳定了不同构象的SgrAI，推测由于活性部位关键残基的重组而被激活。DNA切割动力学表明，在丝状结构形成时，SgrAI对初级和次级位点序列的DNA裂解活性激活了200-1000倍。全程动力学分析表明，SgrAI与细丝的结合是一个缓慢的二级缔合步骤，然后是快速的DNA切割，然后是SgrAI从细丝上解离，最后是快速释放产物(即被切割的DNA)。慢结合步骤控制SgrAI的活性，使其优先在与初级位点序列相连的DNA上形成丝状结构，而将次级位点序列掺入丝状结构中增加了连续DNA上的DNA裂解数量。因此，该系统允许快速切割入侵的噬菌体DNA(具有不受保护的初级位点)。SgrAI系统使用丝状形式的形成来稳定激活的构象。DNA序列和缓慢的二级缔合动力学控制这种细丝形成的地点和时间，其方式经过调整，以实现其生物角色的最佳性能。支持或资助信息这项工作得到了国家科学基金的资助。Mcb-1410355(至N.H.).1Polley等人(2019年)“DNA的间接读出控制序列特异性核酸内切酶的丝化和激活”，Structure 27，1-11.2Barahona等人(2019年)“对速度的需求：连续形成寡聚纤维提供最大速度和最大活性隔离”，J.Virol，93(5)，e01647-18。
SgrAI is allosteric and is both activated as well as undergoes a change in substrate specificity (cleaving a secondary class of DNA sequences) upon binding to its primary recognition sequence in DNA. We have investigated the allosteric activation of SgrAI and this unusual expansion of DNA sequence specificity using structural, biophysical, and enzyme kinetic methods. 1– 2A variety of biophysical techniques (native gel electrophoresis, analytical ultracentrifugation, ion mobility native mass spectrometry, x‐ray crystallography and electron microscopy) to investigate complexes formed by SgrAI. Single turnover DNA cleavage assays were used to investigate the reaction mechanism of SgrAI, and global modeling to create a computation model of the full reaction pathway including rate constants for each important step. The biophysical characterizations revealed that SgrAI forms run‐on oligomers or filaments when activated, and that these filaments are induced when SgrAI binds to its primary recognition sequence. SgrAI binds tightly to secondary site sequences, which differ from primary by a single base pair, but fails to cleave them and does not induce filament formation. However, SgrAI bound to the secondary site sequences will join filaments formed from SgrAI bound to primary site sequences. Protein‐protein and protein‐DNA contacts between SgrAI/DNA complexes in a filament stabilize a different conformation of SgrAI, hypothesized to be activated due to a reorganization of critical residues in the active site. DNA cleavage kinetics reveal activation of DNA cleavage activity by SgrAI of 200–1000 fold for primary and secondary site sequences upon filament formation. Full global kinetic analysis reveals a slow second order association step of SgrAI addition to a filament, followed by rapid DNA cleavage, then dissociation of SgrAI from the filament, and finally rapid product (i.e.cleaved DNA) release. The slow association step controls SgrAI activity to form filaments preferentially on DNA contiguous with primary site sequences, and incorporation of secondary site sequences into the filament increases the number of DNA cleavages on the contiguous DNA. Hence, this system allows for rapid cleavage of invading phage DNA (with unprotected primary sites).The SgrAI system uses formation of a filamentous form to stabilize an activated conformation. DNA sequence and slow second order association kinetics control where and when this filament is formed in a way that is tuned for optimal performance of its biological role.Support or Funding InformationThis work was supported by the National Science Foundation under Grant No. MCB‐1410355 (to N.H.).1Polley, et al ( 2019) “ Indirect Readout of DNA Controls Filamentation and Activation of a Sequence-Specific Endonuclease”, Structure 27, 1– 11.2Barahona, et al ( 2019) “ The Need for Speed: Run-On Oligomer Filament formation provides Maximum Speed with Maximum Sequestration of Activity”, J. Virol, 93 ( 5), e01647– 18.
DOI：
10.1101/585943
发表时间：
2019-03
期刊：
bioRxiv
影响因子：
--
作者：
Smarajit Polley;D. Lyumkis;N. Horton
通讯作者：
Smarajit Polley;D. Lyumkis;N. Horton