RECOGNITION AND CLEAVAGE BY RESTRICTION ENDONUCLEASES

限制性核酸内切酶的识别和切割

• 批准号: 6178906
• 负责人: EVA S VANAMEE
• 金额: $ 4.09万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-05-31 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6178906
• 关键词: DNA; DNA footprinting; Flavobacteriaceae; X ray crystallography; analytical ultracentrifugation; chemical cleavage; crystallization; enzyme mechanism; enzyme structure; fluorescence resonance energy transfer; intermolecular interaction; restriction endonucleases; structural biology

Understanding protein-DNA interaction is essential in the fight of diseases at the genetic level. Restriction endonucleases are excellent model systems to study protein-DNA interaction because of their high specificity towards their recognition sequence. They are part of restriction-modification systems and serve as a primitive immune system in bacterial cells against bacteriophage infection by cutting the phage DNA with high specificity, while the host DNA is protected by a specific methylation within the recognition sequence. Additionally, the high specificity gives restriction endonucleases an important role in recombinant DNA technologies. I propose to study the structure-function of the FokI restriction endonuclease using a combination of biophysical methods. FokI belongs to a special subgroup of bipartite enzymes that cleave DNA nonspecifically at a defined distance outside of a non- palindromic recognition sequence. The bipartite nature of FoAl makes it an excellent candidate for the design of artificial enzymes. The studies will provide further understanding how recognition and catalysis are coupled (or de-coupled) in restriction endonucleases and will aid the design of restriction enzymes with new specificities.

了解蛋白质-DNA相互作用对于在遗传水平上对抗疾病至关重要。限制性核酸内切酶是研究蛋白质-DNA相互作用的理想模型系统，因为它们对其识别序列具有高度的特异性。它们是限制修饰系统的一部分，并通过以高特异性切割噬菌体DNA而在细菌细胞中作为抵抗噬菌体感染的原始免疫系统，同时宿主DNA受到识别序列内的特异性甲基化的保护。此外，高特异性使限制性内切核酸酶在重组DNA技术中发挥重要作用。我建议使用生物物理方法的组合来研究FokI限制性内切酶的结构-功能。FokI属于一种特殊的二分酶亚组，其在非回文识别序列外的限定距离处非特异性地切割DNA。FoAl的二分性质使其成为人工酶设计的优秀候选者。这些研究将提供进一步了解如何识别和催化耦合（或解耦合）的限制性内切酶，并将有助于设计新的限制性内切酶的特异性。