Engineering DNA Endonuclease Reagents for Gene Targeting

工程 DNA 核酸内切酶试剂用于基因打靶

• 批准号: 7122083
• 负责人: FREDERICK Samuel GIMBLE
• 金额: $ 21.34万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-03 至 2009-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7122083
• 关键词: Affect; Architecture; Base Pairing; Binding; Cancer Etiology; Cell Survival; Cells; Characteristics; Class; Cleaved cell; Collaborations; Communities; Complex; Coupled; DNA; DNA Binding; DNA Repair; DNA Sequence; Data; Deoxyribonuclease I; Development; Disease; Engineering; Enzymes; Event; Expression Library; Family; Gene Mutation; Gene Targeting; Gene Transfer; Generations; Genes; Genetic; Genome; Genomics; Goals; Health; Homing; Human; Human Genome; Individual; Introns; Knowledge; Ligands; Light; Methods; Molecular; Mutation; Numbers; Outcome; Positioning Attribute; Proteins; Protocols documentation; Randomized; Reagent; Regulation; Research; Research Personnel; Roentgen Rays; Saccharomyces cerevisiae Proteins; Scientist; Sirolimus; Specificity; Staging; Stimulus; Structure; System; Time; Tissues; Two-Hybrid System Techniques; Variant; Work; Yeasts; base; endodeoxyribonuclease SceI; endonuclease; enzyme activity; experience; genetic selection; homologous recombination; human disease; in vivo; innovation; irradiation; member; mutant; novel; repaired; response; small molecule

DESCRIPTION (provided by applicant): The long-term objective of this project is to develop novel homing endonucleases that can be used in gene targeting protocols to repair the genetic mutations that cause cancer and other human diseases. Homing endonucleases initiate gene targeting by generating double-strand breaks at defined genomic loci that greatly stimulate DNA repair through homologous recombination. The I-Scel protein from S. cerevisiae is a member of the LAGLIDADG family of homing endonucleases that is capable of finding and cleaving a single 18 base-pair recognition sequence within several megabases of non-specific DNA. It has become the reagent-of-choice for studying DNA repair and for initiating gene targeting because of its extreme specificity, which limits DNA cleavage at ectopic targets that negatively impact cell viability. This proposal expands the utility of I-Scel by more precisely controlling its activity in vivo and by evolving the enzyme to specifically recognize other DNA targets. The recent determination of the X-ray structure of I-Scel reveals a two-domain topology that permits the re-engineering of the enzyme. The first Specific Aim engineers molecular switches into the enzyme that allow the activity to be temporally and spatially regulated. Fusion of the I-Scel domains to different protein partners will create one endonuclease whose activity is triggered by the small-molecule rapamycin and one that can be reversibly switched on and off in response to light. The generation of these enzymes will permit the first in vivo regulation of the DNA repair activity and will allow repair events to be studied in single cells. The second Specific Aim uses a bacterial two-hybrid method to alter the DNA target specificity of I-Scel. Individual variants will be selected from a large randomized I-Scel expression library that gain the ability to bind to a mutant recognition sequence but lose the wild-type target specificity. These studies will ultimately increase the repertoire of homing endonucleases available for gene targeting.

描述（由申请人提供）：该项目的长期目标是开发新的归巢核酸内切酶，这些内切核酸酶可用于基因靶向方案，以修复引起癌症和其他人类疾病的遗传突变。归巢核酸内切酶通过在定义的基因组基因座上产生双链断裂，从而启动基因靶向，从而通过同源重组极大地刺激DNA修复。酿酒酵母的I-Scel蛋白是洛格里达格家族的归核核酸酶的成员，该家族能够在几个非特异性DNA的兆千个兆座中找到和裂解单个18碱基识别序列。它已成为研究DNA修复和启动基因靶向的选择试剂，因为它的极端特异性限制了异位靶标的DNA裂解，从而对细胞的生存能力产生负面影响。该提案通过更精确地控制其在体内活性和发展酶以明确识别其他DNA靶标，从而扩展了I-SCEL的实用性。 i-Scel的X射线结构的最新确定揭示了允许重新设计酶的两域拓扑。 第一个特定的目标工程师分子开关进入酶，使活性受到时间和空间调节。 I-SCEL域与不同蛋白质伴侣的融合将产生一种核酸酶，其活性是由小分子雷帕霉素触发的，并且可以响应光线而被可逆地打开和关闭。这些酶的产生将允许对DNA修复活性的第一个体内调节，并允许在单个细胞中研究修复事件。第二个特定目的使用细菌两性杂交方法来改变I-SCEL的DNA靶特异性。单个变体将从大型的随机I-Scel表达式库中选择，该库获得与突变体识别序列结合但失去野生型目标特异性的能力。这些研究最终将增加可用于基因靶向的归巢核酸内切酶的曲目。