RECOGNITION AND CLEAVAGE BY RESTRICTION ENDONUCLEASES

限制性核酸内切酶的识别和切割

• 批准号: 6385056
• 负责人: EVA S VANAMEE
• 金额: $ 4.56万
• 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/6385056
• 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 相互作用对于在基因水平上对抗疾病至关重要。限制性核酸内切酶因其识别序列的高度特异性而成为研究蛋白质-DNA 相互作用的优秀模型系统。它们是限制性修饰系统的一部分，通过高特异性切割噬菌体 DNA，充当细菌细胞中对抗噬菌体感染的原始免疫系统，而宿主 DNA 则受到识别序列内特定甲基化的保护。此外，高特异性使限制性内切酶在重组 DNA 技术中发挥重要作用。我建议结合生物物理方法来研究 FokI 限制性内切酶的结构功能。 FokI 属于二分酶的特殊亚组，其在非回文识别序列之外的规定距离处非特异性切割 DNA。 FoAl 的二分性质使其成为人工酶设计的绝佳候选者。这些研究将进一步了解限制性内切酶中识别和催化如何耦合（或解耦合），并将有助于设计具有新特异性的限制性酶。