Identification of a conserved DNA sulfur recognition domain by characterizing the phosphorothioate-specific endonuclease SprMcrA from Streptomyces pristinaespiralis
Identification of a conserved DNA sulfur recognition domain by characterizing the phosphorothioate-specific endonuclease SprMcrA from Streptomyces pristinaespiralis
复制标题
通过表征原始螺旋链霉菌的硫代磷酸酯特异性核酸内切酶 SprMcrA 来鉴定保守的 DNA 硫识别结构域
DOI：
10.1111/mmi.14118
复制
发表时间：
2018-11-01
影响因子：
3.6
通讯作者：
He, Xinyi
中科院分区：
文献类型：
--
作者：
Yu, Hao;Liu, Guang;He, Xinyi
链霉菌是理解DNA硫代磷酸化现象的有价值的模型，其中硫取代了DNA磷酸骨架中的非桥氧。我们以前报道过天蓝色链霉菌的限制性内切酶ScoMcrA在距离修饰位点16-28个核苷酸的位点切割硫代磷酸DNA和Dcm甲基化DNA。然而，ScoMcrA对修饰的DNA的切割总是不完全的，并且伴随着对未修饰的DNA的严重混杂活性。这些特征使硫代磷酸酯DNA的识别和切割的研究复杂化。出于这些原因，我们在这里的特点SprMcrA从Streptomyces pristinaespiralis，一个小得多的同源物ScoMcrA与罕见的HRH基序，HNH基序的变体，形成这些内切酶的催化中心。确定了SprMcrA的硫结合结构域及其硫代磷酸化识别位点。与ScoMcrA相比，SprMcrA在从未修饰的DNA中辨别硫代磷酸酯DNA方面具有更高的特异性，并且这种酶通常在距离识别位点的5 '侧11-14个核苷酸的距离处切割两条链。HRH/HNH基序在DNA水解中具有其自身的序列特异性，导致在某些硫代磷酸化位点的切割失败。HRH中心残基的R248 N突变导致硫代磷酸DNA的切割活性增强30倍，并改变了某些位点的切割效率，而两个His残基的突变废除了限制活性。这是第一次报告的识别结构域的硫代磷酸DNA和硫代磷酸依赖性和序列特异性限制活性。
Streptomyces species have been valuable models for understanding the phenomenon of DNA phosphorothioation in which sulfur replaces a non-bridging oxygen in the phosphate backbone of DNA. We previously reported that the restriction endonuclease ScoMcrA from Streptomyces coelicolor cleaves phosphorothioate DNA and Dcm-methylated DNA at sites 16-28 nucleotides away from the modification sites. However, cleavage of modified DNA by ScoMcrA is always incomplete and accompanied by severe promiscuous activity on unmodified DNA. These features complicate the studies of recognition and cleavage of phosphorothioate DNA. For these reasons, we here characterized SprMcrA from Streptomyces pristinaespiralis, a much smaller homolog of ScoMcrA with a rare HRH motif, a variant of the HNH motif that forms the catalytic center of these endonucleases. The sulfur-binding domain of SprMcrA and its phosphorothioation recognition site were determined. Compared to ScoMcrA, SprMcrA has higher specificity in discerning phosphorothioate DNA from unmodified DNA, and this enzyme generally cuts both strands at a distance of 11-14 nucleotides from the 5 ' side of the recognition site. The HRH/HNH motif has its own sequence specificity in DNA hydrolysis, leading to failure of cleavage at some phosphorothioated sites. An R248N mutation of the central residue in HRH resulted in 30-fold enhancement in cleavage activity of phosphorothioate DNA and altered the cleavage efficiency at some sites, whereas mutation of both His residues abolished restriction activity. This is the first report of a recognition domain for phosphorothioate DNA and phosphorothioate-dependent and sequence-specific restriction activity.