Assembly of the Bacteriophage T4 DNA Replication Sliding Clamp: A Functional Analysis of Protein-Protein Subassemblies

噬菌体 T4 DNA 复制滑动夹的组装：蛋白质-蛋白质亚组装体的功能分析

• 批准号: 9506139
• 负责人: Michael Reddy
• 金额: $ 37万
• 依托单位: University of Wisconsin-Milwaukee
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-15 至 2001-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9506139&HistoricalAwards=false
• 关键词: Assembly; Bacteriophage; T4; DNA; Replication

9506139 Reddy This research will determine the key steps by which a multicomponent complex involved in the replication bacteriophage T4 DNA is assembled. In particular, this research will yield a precise understanding of how complex termed the "clamp loader" (gp44/62, a hetero-oligomeric protein possessing an intrinsic ATPase activity) specifically interacts with a second protein, termed the "sliding clamp" (gp45, a homo-oligomeric protein) to subsequently "load" the sliding clamp onto DNA. A variety of inter- and intracrosslinking methodologies, including both chemical as well as molecular biological approaches will be employed. Using a recently refined version of the nitrocellulose filter binding assay, a thorough kinetic analysis of the binding and release of ADP to the ATP-dependent clamp loader protein complex will be done. Development of novel protein chromatographic schemes such as affinity chromatography in the presence of a macromolecular crowding agent (polyethylene glycol) or specific retention of protein-protein complexes by the inclusion of Mg-ATP in all chromatographic buffers will be explored in order to facilitate the purification of intact protein-protein complexes directly from biological sources. The nature of the macromolecular interactions observed in the T4 system appear to closely parallel those currently under investigation in the field of molecular chaperones. Therefore, insights garnered from these studies will add to the growing understanding of the molecular basis underlying the specificity and regulation of protein-protein interactions. Current avenues of investigations in molecular and cellular biochemistry will be presented to both undergraduate and graduate students by a research-level analysis (lectures and assignments) in which understanding is considerably valued over rote memorization. Two "Special Topics in Biochemistry" courses entitled "The Enzymology of DNA Replication" and Cells as Macromolecular Assemblies" will provide students with a breath of knowledge that enables them to independently think about molecular mechanisms of biological relevance. These courses combine formal lectures with class-sessions during which there is a critical assessment of directly relevant papers assigned from the primary scientific literature. Modernization of a general biochemistry course will be accomplished by complementing molecular biology lectures with student assignments such as searching for DNA sequence homologies, designing oligonucleohdes for use in PCR, and performing secondary structure predictions. These tasks will require of the students that they learn how to navigate, using computers, the Internet (in particular, the World Wide Web). An undergraduate Experimental Biochemistry Laboratory course has been designed as a mini-research project around the enzyme bacterial luciferase. A laboratory manual also has been written for this course. The structure of the course provides the students with an excellent and exciting model system by which to learn various common and important techniques in biochemistry and molecular biology. %%% This research will determine the key steps by which a multiprotein complex involved in the replication of bacteriophage T4 DNA is assembled. Crucial features of the mechanism of DNA replication are conserved throughout Nature. Therefore, insights garnered from these studies will add to the growing understanding of the molecular basis underlying the specificity and regulation of protein-protein interactions in general and of DNA replication in particular. Current avenues of investigations in molecular and cellular biochemistry will be presented to both undergraduate and graduate students by a research-level analysis (lectures and assignments) in which understanding is considerably valued over rote memorization. Two "Special Topics in Biochemistry" courses entitled "The Enzymology of DNA Replication" and Cells as Macromolecular Assemblies" will provide students with a breath of knowledge that enables them to ind ependently think about molecular mechanisms of biological relevance. These courses combine formal lectures with class-sessions during which there is a critical assessment of directly relevant papers assigned from the primary scientific literature. Modernization of a general biochemistry course will be accomplished by complementing molecular biology lectures with student assignments such as searching for DNA sequence homologies, designing oligonucleohdes for use in PCR, and performing secondary structure predictions. These tasks will require of the students that they learn how to navigate, using computers, the Internet (in particular, the World Wide Web). An undergraduate Experimental Biochemistry Laboratory course has been designed as a mini-research project around the enzyme bacterial luciferase. A laboratory manual also has been written for this course. The structure of the course provides the students with an excellent and exciting model system by which to learn various common and important techniques in biochemistry and molecular biology. ***

9506139 Reddy 这项研究将确定参与复制噬菌体 T4 DNA 的多组分复合物的组装关键步骤。 特别是，这项研究将准确地理解复杂的“夹钳加载器”（gp44/62，一种具有内在 ATP 酶活性的异源寡聚蛋白）如何与第二种蛋白质（称为“滑动夹”（gp45，一种同源寡聚蛋白））特异性相互作用，从而随后将滑动夹“加载”到 DNA 上。 将采用各种内部和内部交联方法，包括化学和分子生物学方法。 使用最近改进的硝酸纤维素滤膜结合测定法，将对 ADP 与 ATP 依赖性夹钳装载蛋白复合物的结合和释放进行彻底的动力学分析。 将探索新型蛋白质色谱方案的开发，例如在大分子拥挤剂（聚乙二醇）存在下的亲和色谱或通过在所有色谱缓冲液中包含 Mg-ATP 来特异性保留蛋白质-蛋白质复合物，以促进直接从生物来源纯化完整的蛋白质-蛋白质复合物。 T4 系统中观察到的大分子相互作用的性质似乎与目前分子伴侣领域正在研究的大分子相互作用的性质非常相似。 因此，从这些研究中获得的见解将有助于加深对蛋白质-蛋白质相互作用的特异性和调节的分子基础的理解。 当前分子和细胞生物化学的研究途径将通过研究水平的分析（讲座和作业）向本科生和研究生展示，其中理解比死记硬背更重要。两门“生物化学专题”课程，题为“DNA 复制的酶学”和“细胞作为大分子组装体”，将为学生提供知识气息，使他们能够独立思考生物相关的分子机制。这些课程将正式讲座与课堂教学相结合，课堂教学对从主要科学文献中分配的直接相关论文进行严格评估。 生物化学课程将通过补充分子生物学讲座和学生作业来完成，例如搜索 DNA 序列同源性、设计用于 PCR 的寡核苷酸以及进行二级结构预测。这些任务要求学生学习如何使用计算机和互联网（特别是万维网）进行导航。 本科生实验生物化学实验室课程被设计为 围绕细菌荧光素酶的小型研究项目。 本课程还编写了实验室手册。 课程结构为学生提供了一个优秀且令人兴奋的模型系统，通过该模型系统学习生物化学和分子生物学中的各种常见和重要技术。 %%% 这项研究将确定参与噬菌体 T4 DNA 复制的多蛋白复合物组装的关键步骤。 该机制的关键特征 DNA 复制的过程在自然界中是保守的。 因此，从这些研究中获得的见解将有助于加深对蛋白质-蛋白质相互作用、特别是 DNA 复制的特异性和调节的分子基础的理解。 当前分子和细胞生物化学的研究途径将通过研究水平的分析（讲座和作业）向本科生和研究生展示，其中理解非常重要 比死记硬背更有价值。两门“生物化学专题”课程，题为“DNA复制的酶学”和“细胞作为大分子组装体”，将为学生提供丰富的知识，使他们能够独立思考生物相关的分子机制。 这些课程将正式讲座与课堂会议相结合，期间对主要科学文献中分配的直接相关论文进行严格评估。 一般生物化学课程的现代化将通过补充分子生物学讲座和学生作业来完成，例如搜索 DNA 序列同源性、设计用于 PCR 的寡核苷酸以及执行二级结构预测。这些任务要求学生学习如何使用计算机和互联网（特别是万维网）进行导航。 本科生实验生物化学实验室课程被设计为围绕细菌荧光素酶的小型研究项目。 本课程还编写了实验室手册。 课程结构为学生提供了一个优秀且令人兴奋的模型系统，通过该模型系统学习生物化学和分子生物学中的各种常见和重要技术。 ***