RECOGNITION AND CLEAVAGE BY RESTRICTION ENDONUCLEASES

限制性核酸内切酶的识别和切割

• 批准号: 2862880
• 负责人: EVA S VANAMEE
• 金额: $ 3.84万
• 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/2862880
• 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.

了解蛋白质与DNA的相互作用对于在基因水平上与疾病作斗争至关重要。限制性内切酶对其识别序列具有高度的特异性，是研究蛋白质-DNA相互作用的优秀模型系统。它们是限制性内切酶修饰系统的一部分，在细菌细胞中作为原始免疫系统，通过以高特异性切割噬菌体DNA来抵御噬菌体感染，而宿主DNA则受到识别序列中特定甲基化的保护。此外，高度的特异性使限制性内切酶在重组DNA技术中发挥着重要作用。我建议结合生物物理方法来研究FokI限制性内切酶的结构和功能。FokI属于两部分酶的一个特殊亚群，它们在非回文识别序列之外的特定距离非特异性地切割DNA。马驹的两部分性质使其成为设计人造酶的极佳候选者。这些研究将进一步了解限制性内切酶中识别和催化是如何耦合(或解偶联)的，并将有助于设计具有新特性的限制性内切酶。