Effects of DNA Binding of Zinc Finger and Linkers for Domain Fusion on Catalytic Activity of Sequence-Specific Chimeric Recombinases Determined by a Facile Fluorescent System
Effects of DNA Binding of Zinc Finger and Linkers for Domain Fusion on Catalytic Activity of Sequence-Specific Chimeric Recombinases Determined by a Facile Fluorescent System
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锌指和连接体的 DNA 结合对结构域融合对通过简易荧光系统测定的序列特异性嵌合重组酶催化活性的影响
作者：
Nomura;W.;Masuda;A.;Ohba;K.;Urabe;A.;Ito;N.;Ryo;A.;Yamamoto;N.;Tamamura;H.
人工锌指蛋白（ZFP）由Cys 2-His 2型模块组成，该模块由130个氨基酸组成，具有与锌离子配位的ββα结构。识别特定DNA靶序列的ZFP可以取代作用于DNA的酶的结合结构域，以产生具有可编程序列特异性的设计酶。这些工程酶中研究最多的是锌指核酸酶（ZFN）。ZFN已被广泛用于模拟生物体，目前正在进行人类临床试验，目的是进行治疗性基因编辑。ZFN的困难来自非同源末端连接和脱靶DNA切割和诱变引起的不可预测的突变。旨在解决ZFN的缺点的更近期的策略涉及锌指重组酶（ZFR）。对ZFR及其修饰方法的深入了解有望为模式生物中的基因操作以及基因治疗提供强大的新工具。在设计有效和特异性ZFR的努力中，已经评估了锌指结构域和ZFP与重组酶催化结构域之间的接头序列的DNA结合亲和力的影响。构建了含有ZFR靶位点的质粒系统，用于评价具有可变接头长度和锌指模块数目的ZFR的催化活性。通过对大肠杆菌中反应后分离质粒的限制性内切酶分析和哺乳动物细胞中EGFP荧光的变化来评估纯化效率。这些结果提供了与ZFR的设计相关的信息，这些ZFR将用于序列特异性基因组修饰。
Artificial zinc finger proteins (ZFPs) consist of Cys2-His2-type modules composed of ∼30 amino acids with a ββα structure that coordinates a zinc ion. ZFPs that recognize specific DNA target sequences can substitute for the binding domains of enzymes that act on DNA to create designer enzymes with programmable sequence specificity. The most studied of these engineered enzymes are zinc finger nucleases (ZFNs). ZFNs have been widely used to model organisms and are currently in human clinical trials with an aim of therapeutic gene editing. Difficulties with ZFNs arise from unpredictable mutations caused by nonhomologous end joining and off-target DNA cleavage and mutagenesis. A more recent strategy that aims to address the shortcomings of ZFNs involves zinc finger recombinases (ZFRs). A thorough understanding of ZFRs and methods for their modification promises powerful new tools for gene manipulation in model organisms as well as in gene therapy. In an effort to design efficient and specific ZFRs, the effects of the DNA binding affinity of the zinc finger domains and the linker sequence between ZFPs and recombinase catalytic domains have been assessed. A plasmid system containing ZFR target sites was constructed for evaluation of catalytic activities of ZFRs with variable linker lengths and numbers of zinc finger modules. Recombination efficiencies were evaluated by restriction enzyme analysis of isolated plasmids after reaction inEscherichia coliand changes in EGFP fluorescence in mammalian cells. The results provide information relevant to the design of ZFRs that will be useful for sequence-specific genome modification.