Abstract：
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.
摘要：
酶聚合(也称为成丝)已经作为酶调节的新的层出现。SgrAI是序列依赖性DNA内切核酸酶,其形成具有增强的DNA切割活性以及改变的DNA序列特异性的聚合物丝。为了更好地理解这种不寻常的监管机制，反应途径的完整全局动力学建模，包括酶丝化步骤，已经进行了。通过SgrAI切割的初级DNA识别序列的先前工作已经显示了如何调节每个反应步骤的动力学速率常数以最大化活化和DNA切割,同时最小化DNA切割到宿主基因组的程度。在目前的工作中,我们扩展了我们之前的研究，现在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的成丝和激活。这项工作表明，低活性状态和高活性状态之间的变构平衡受到结合DNA序列的调节，初级序列更容易激活和细丝形成，而与二级识别序列结合的SgrAI以高达40倍的优势有利于低（和非丝状）状态。此外,宿主生物中二级序列的甲基化程度，灰色链霉菌，现在是第一次报道，并显示正如预测的那样，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纤丝形成酶中至关重要。
