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

Reddy：这项研究将确定参与噬菌体T4 DNA复制的多组分复合体组装的关键步骤。特别是，这项研究将产生一个精确的理解，如何复杂的称为“钳加载器”（gp44/62，一种具有内在atp酶活性的异寡聚蛋白）特异性地与第二种蛋白质，称为“滑动钳”（gp45，一种同源寡聚蛋白）相互作用，随后将滑动钳“加载”到DNA上。各种交联和内交联方法，包括化学和分子生物学方法将被采用。使用最近改进的硝化纤维素过滤器结合试验，将对ADP与atp依赖性钳装器蛋白复合物的结合和释放进行彻底的动力学分析。新的蛋白质色谱方案的发展，如亲和层析存在大分子拥挤剂（聚乙二醇）或特定保留蛋白-蛋白复合物通过在所有色谱缓冲液中包含Mg-ATP将被探索，以促进纯化完整的蛋白质-蛋白质复合物直接从生物来源。在T4系统中观察到的大分子相互作用的性质似乎与目前在分子伴侣领域的研究密切相似。因此，从这些研究中获得的见解将增加对蛋白质-蛋白质相互作用的特异性和调节的分子基础的日益增长的理解。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）呈现给本科生和研究生，在这种分析中，理解比死记硬背更重要。两个“生物化学专题”课程“DNA复制的酶学”和“细胞作为大分子组装”将为学生提供知识的呼吸，使他们能够独立思考生物学相关的分子机制。这些课程结合了正式的讲座和课堂教学，在课堂教学中，对主要科学文献中指定的直接相关论文进行批判性评估。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，例如寻找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。这些任务将要求学生学会如何使用计算机浏览互联网（特别是万维网）。本科实验生物化学实验课程是围绕细菌荧光素酶设计的一个小型研究项目。本课程还编写了一份实验手册。本课程的结构为学生提供了一个优秀的和令人兴奋的模型系统，通过它来学习生物化学和分子生物学中各种常见和重要的技术。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。DNA复制机制的关键特征在自然界中是保守的。因此，从这些研究中获得的见解将增加对蛋白质-蛋白质相互作用的特异性和调控的分子基础的理解，特别是DNA复制。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）呈现给本科生和研究生，在这种分析中，理解比死记硬背更重要。两个“生物化学专题”课程，“DNA复制的酶学”和“细胞作为大分子组装”将为学生提供知识的呼吸，使他们能够独立思考生物学相关的分子机制。这些课程结合了正式的讲座和课堂教学，在课堂教学中，对主要科学文献中指定的直接相关论文进行批判性评估。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，例如寻找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。这些任务将要求学生学会如何使用计算机浏览互联网（特别是万维网）。本科实验生物化学实验课程是围绕细菌荧光素酶设计的一个小型研究项目。本课程还编写了一份实验手册。本课程的结构为学生提供了一个优秀的和令人兴奋的模型系统，通过它来学习生物化学和分子生物学中各种常见和重要的技术。* * *

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. ***