Structural studies of Sleeping Beauty transposase

描述(申请人提供)：DNA转座子是离散的DNA序列，可以从基因组的一个位置移动(转座)到另一个位置。它们几乎存在于所有生物体中，对基因组结构和功能都有贡献。DNA转座子也是一种天然的基因传递工具，正在被开发为基因组工程的工具，如插入突变和转基因，以及用于人类基因治疗。DNA转座反应发生在蛋白质-DNA复合体(转座体)内，转座体包含两个或两个以上的转座酶和转座子DNA的末端。尽管利用生物化学和分子遗传学的方法在理解DNA转座方面取得了重大进展，但关于转座体组装和操作的功能和结构机制的基本问题仍然没有得到解决。我的实验室的长期目标是阐明这些机制，以及如何将它们用于不同的遗传应用。在这个项目中，我们建议研究睡美人(SB)DNA转座子，这是在研究遗传学应用中最广泛使用的转座子，也是第一个也是唯一一个用于人类基因治疗临床试验的DNA转座子。这项建议的具体目标是获得急需的关于SB转座酶及其与DNA结合的高分辨率结构和动力学信息。我们的实验方法集成了先进的溶液核磁共振技术、诱变和生化功能分析。这一建议的具体目标是：(1)确定Sb转座酶的关键功能重要结构特征；(2)Sb转座酶的功能动力学；(3)确定Sb转座酶的红色亚域在转座子DNA识别中的作用。这些结果将对了解DNA转座酶的功能、结构和动力学特征以及利用SB转座子进行人类基因治疗和向动物细胞运送基因具有重要的特殊应用。

DESCRIPTION (provided by applicant): DNA transposons are discrete DNA sequences that can move (transpose) from one location in the genome to another. They are present in virtually all organisms and contribute to both genome structure and function. DNA transposons are also natural gene delivery vehicles that are being developed as tools for genome engineering, such as insertional mutagenesis and transgenesis, and for human gene therapy. The reaction of DNA transposition occurs within a protein-DNA complex (transpososome), which contains two or more transposase enzymes and the ends of the transposon DNA. Despite the significant progress in understanding the DNA transposition has been made using biochemical and molecular genetics approaches, fundamental questions about the functional and structural mechanisms underpinning transpososome assembly and operation remain unaddressed. The long-term objective of my laboratory is to elucidate these mechanisms and how they can be exploited for different genetic applications. In this project we propose to investigate a Sleeping Beauty (SB) DNA transposon, the most widely used transposon in research genetic applications and the first and only DNA transposon in clinical trials for human gene therapy. The specific objective of this proposal is to obtain the critically needed high-resolution structural and dynamics information on SB transposase and its binding to DNA. Our experimental approach integrates advanced solution NMR techniques, mutagenesis, and biochemical functional assays. The specific aims of this proposal are directed at (1) identifying key functionally important structural features of the SB transposase, (2) the functional dynamics of the SB transposase, and (3) establishing the role of the RED subdomain of SB transposase in the transposon DNA recognition. The results will have broad applications to understanding the functional structural and dynamics features of DNA transposases and significant particular applications to human gene therapy and gene delivery to animal cells using the SB transposon.