Enzyme polymerization (also known as filamentation) has emerged as a new layer of enzyme regulation. SgrAI is a sequence-dependent DNA endonuclease that forms polymeric filaments with enhanced DNA cleavage activity as well as altered DNA sequence specificity. To better understand this unusual regulatory mechanism, full global kinetic modeling of the reaction pathway, including the enzyme filamentation steps, has been undertaken. Prior work with the primary DNA recognition sequence cleaved by SgrAI has shown how the kinetic rate constants of each reaction step are tuned to maximize activation and DNA cleavage while minimizing the extent of DNA cleavage to the host genome. In the current work, we expand on our prior study by now including DNA cleavage of a secondary recognition sequence, to understand how the sequence of the bound DNA modulates filamentation and activation of SgrAI. The work shows that an allosteric equilibrium between low and high activity states is modulated by the sequence of bound DNA, with primary sequences more prone to activation and filament formation, while SgrAI bound to secondary recognition sequences favor the low (and nonfilamenting) state by up to 40-fold. In addition, the degree of methylation of secondary sequences in the host organism, Streptomyces griseus, is now reported for the first time and shows that as predicted, these sequences are left unprotected from the SgrAI endonuclease making sequence specificity critical in this unusual filament-forming enzyme.

中文翻译：

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酶丝形成的 DNA 序列控制和 SgrAI 核酸内切酶的激活

酶聚合（也称为丝化）已成为酶调节的新层。 SgrAI 是一种序列依赖性 DNA 核酸内切酶，可形成具有增强的 DNA 切割活性以及改变的 DNA 序列特异性的聚合丝。为了更好地理解这种不寻常的调节机制，我们对反应途径（包括酶丝化步骤）进行了完整的全局动力学建模。先前对 SgrAI 切割的初级 DNA 识别序列的研究表明，如何调整每个反应步骤的动力学速率常数以最大化激活和 DNA 切割，同时最小化 DNA 对宿主基因组的切割程度。在当前的工作中，我们扩展了之前的研究，包括二级识别序列的 DNA 切割，以了解结合 DNA 的序列如何调节 SgrAI 的丝化和激活。这项工作表明，低活性状态和高活性状态之间的变构平衡是由结合的 DNA 序列调节的，初级序列更容易激活和丝状形成，而与二级识别序列结合的 SgrAI 有利于低（和非丝状）状态至40倍。此外，首次报道了宿主生物体灰色链霉菌中二级序列的甲基化程度，并表明正如预测的那样，这些序列不受 SgrAI 核酸内切酶的保护，使得序列特异性在这种不寻常的丝状形成中至关重要酶。