Indirect Readout of DNA Controls Filamentation and Activation of a Sequence-Specific Endonuclease
Indirect Readout of DNA Controls Filamentation and Activation of a Sequence-Specific Endonuclease
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DOI：
10.1101/585943
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发表时间：
2019-03
期刊：
影响因子：
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通讯作者：
Smarajit Polley;D. Lyumkis;N. Horton
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文献类型：
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作者：
Smarajit Polley;D. Lyumkis;N. Horton
由酶形成的丝状或连续型低聚物越来越被认为是一种重要的现象，具有潜在的独特的调节特性和生物学作用。SgrAI是一种变构调控的II型限制性内切酶，它能形成连续型寡聚体(Roo)丝，具有增强的DNA切割活性和改变的序列特异性。在这里，我们展示了SgrAI的Roo细丝与切割的初级DNA和Mg2+的模拟物结合的3.5？低温电子显微镜结构。较大的构象变化稳定了催化口袋内的第二个金属离子辅因子结合部位，并有助于组装能够快速切割DNA的高级酶形式。这些结构变化阐明了丝状SgrAI形式中超加速DNA切割活动的机制起源。对蛋白质-DNA界面和单个核苷酸的堆积的分析揭示了丝状SgrAI中的间接DNA读出如何能够识别比其低活性形式多得多的核苷酸序列，从而扩大了DNA序列的特异性。总之，底物DNA结合、间接读出和丝状化同时增强了SgrAI的催化活性并调节了底物的选择性。这种不寻常的酶机制可能已经进化成执行细菌天然免疫的特殊功能，以快速防御入侵的噬菌体DNA，而不会对宿主DNA造成损害。
Filament or run-on oligomer formation by enzymes is increasingly recognized as an important phenomenon with potentially unique regulatory properties and biological roles. SgrAI is an allosterically regulated type II restriction endonuclease that forms run-on oligomeric (ROO) filaments with enhanced DNA cleavage activity and altered sequence specificity. Here, we present the 3.5 Å cryo-electron microscopy structure of the ROO filament of SgrAI bound to a mimic of cleaved primary site DNA and Mg2+. Large conformational changes stabilize a second metal ion cofactor binding site within the catalytic pocket and facilitate assembling a higher-order enzyme form that is competent for rapid DNA cleavage. The structural changes illuminate the mechanistic origin of hyper-accelerated DNA cleavage activity within the filamentous SgrAI form. An analysis of the protein-DNA interface and the stacking of individual nucleotides reveals how indirect DNA readout within filamentous SgrAI enables recognition of substantially more nucleotide sequences than its low-activity form, thereby expanding DNA sequence specificity. Together, substrate DNA binding, indirect readout, and filamentation simultaneously enhance SgrAI’s catalytic activity and modulate substrate preference. This unusual enzyme mechanism may have evolved to perform the specialized functions of bacterial innate immunity in rapid defense against invading phage DNA without causing damage to the host DNA.