Purification and enzymatic characterization of Gallus gallus BLM helicase
Purification and enzymatic characterization of Gallus gallus BLM helicase
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原鸡 BLM 解旋酶的纯化和酶学表征
DOI：
10.1093/jb/mvx013
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发表时间：
2017
影响因子：
2.7
通讯作者：
Xi Xu-Guang
中科院分区：
文献类型：
--
作者：
Shi Jing;Liu Na-Nv;Yang Yan-Tao;Xi Xu-Guang
人类BLM解旋酶的突变导致常染色体隐性遗传性Bloom综合征，该综合征显示出对各种类型的恶性肿瘤的高易感性。虽然大量的生化和结构研究已经揭示了解旋酶的核心，但由于其稳定性和产量较低，对整个BLM蛋白的结构研究仍然有限。在此，通过与其他BLM同源物的表达系统和功能的比较，我们建立了鸡BLM(GBLM)在大肠杆菌中的异源高效表达和高产纯化系统。随后的DNA结合和解离检测表明，gBLM是一种活力旺盛的非典型DNA结构特异性解旋酶，它不仅对3‘-尾部的DNA结构表现出高度的选择性，而且能有效地解开末端钝化的泡泡DNA结构，表明它在处理DNA代谢中间体方面具有生物学作用。进一步对gBLM和gBLM Core的比较分析表明，长的N-末端结构域促进了分叉和泡状DNA结构的结合，也是gBLM的DNA解离活性所必需的。因此，我们首次提出了gBLM及其N-末端结构域的酶学特性，为探索人BLM的机制和结构提供了一个新的模型。
Mutations in human BLM helicase give rise to the autosomal recessive Bloom syndrome, which shows high predisposition to types of malignant tumours. Though lots of biochemical and structural investigations have shed lights on the helicase core, structural investigations of the whole BLM protein are still limited due to its low stability and production. Here by comparing with the expression systems and functions of other BLM homologues, we developed the heterologous high-level expression and high-yield purification systems forGallus gallusBLM (gBLM) inEscherichia coli. Subsequent DNA binding and unwinding determinations demonstrated that gBLM was a vigorous atypical DNA structure specific helicase, which not only showed high preference for the 3′-tailed DNA structures but also could efficiently unwind bubble DNA structures with blunt-ends, indicating its biological roles in processing DNA metabolism intermediates. Further comparative analysis between gBLM and gBLM Core revealed that the long N-terminal domain facilitated the binding affinity of forked and bubble DNA structures and it was also required for the DNA unwinding activities of gBLM. Thus, we present the first enzymatic characterization of gBLM and its N-terminal domain, providing a new model for probing the mechanism and structure of human BLM.