Structural studies of Sleeping Beauty transposase

睡美人转座酶的结构研究

基本信息

批准号：
8772933
负责人：
Irina V Nesmelova
金额：
$ 32.73万
依托单位：
UNIVERSITY OF NORTH CAROLINA CHARLOTTE
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-08-01 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8772933
关键词：
Accounting Animals B lymphoid malignancy Binding Biochemical Biochemical Genetics Biological Assay Catalytic Domain Cells Clinical Treatment Clinical Trials Complex Crystallization DNA DNA Binding DNA Binding Domain DNA Sequence DNA Transposons Data Disease Enzymes Family Gene Delivery Gene Transfer Techniques Genetic Genetic Research Genome Genome engineering Goals Human Insertional Mutagenesis Integrase Investigation Laboratories Location Modeling Molecular Conformation Molecular Evolution Molecular Genetics Motion Mutagenesis Mutation NMR Spectroscopy Nature Nuclear Magnetic Resonance Organism Physiological Production Property Protein Dynamics Proteins Reaction Resolution Resolvase Ribonuclease H Role Site Sleeping Beauty Solubility Solutions Structure System Techniques Therapeutic Studies Time Transferase Transgenic Animals Transposase Variant Work base biochemical evolution flexibility functional genomics gene therapy insight operation public health relevance three dimensional structure tool

项目摘要

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.

描述（由申请人提供）：DNA 转座子是离散的 DNA 序列，可以从基因组中的一个位置移动（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 转座子也是天然的基因传递载体，正在被开发为基因组工程（例如插入突变和转基因）以及人类基因治疗的工具。 DNA 转座反应发生在蛋白质-DNA 复合物（转座体）内，该复合物包含两个或多个转座酶和转座子 DNA 的末端。尽管利用生化和分子遗传学方法在理解 DNA 转座方面取得了重大进展，但关于支持转座体组装和操作的功能和结构机制的基本问题仍未得到解决。我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，我们计划研究睡美人 (SB) DNA 转座子，它是研究遗传应用中使用最广泛的转座子，也是人类基因治疗临床试验中第一个也是唯一一个 DNA 转座子。该提案的具体目标是获得急需的有关 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、诱变和生化功能测定。该提案的具体目标是（1）确定 SB 转座酶的关键功能性重要结构特征，（2）SB 转座酶的功能动力学，以及（3）建立 SB 转座酶的 RED 亚结构域在转座子 DNA 识别。这些结果将在理解 DNA 转座酶的功能结构和动力学特征方面具有广泛的应用，并且在人类基因治疗和使用 SB 转座子将基因传递到动物细胞方面具有重要的特殊应用。