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

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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. ***