Mechanism and Evolutionary Design of DNA Polymerase Clamp Loaders.

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Project Summary/Abstract Our proposed research seeks to understand the evolutionary origin and capacity to adapt of complex molecular machines. It is challenging to comprehend how protein function, which depends on finely tuned cooperativity between many components, is adapted and altered as an organism evolves. That is, how does evolution satisfy the demands of high performance while maintaining the capacity for diversification and adaptive specialization? To gain insight into these processes we plan to carry out high-throughput mutagenesis and functional studies of a set of proteins involved in DNA replication. High-speed DNA replication relies on proteins known as sliding clamps, which are proteins that encircle DNA and can diffuse rapidly along the double-helix without dissociating from it. Because the sliding clamps form closed circles, they do not readily associate with DNA on their own. Sliding clamps are opened and loaded onto the start sites of DNA replication by ATP-driven molecular machines called clamp loaders. Clamp loaders are members of an evolutionarily ancient family of ATP-dependent molecular machines called AAA+ ATPases, which are a diverse set of proteins that transduce ATP binding and hydrolysis into mechanical action on proteins. Because the DNA polymerase clamp-loaders are very well understood in terms of their three- dimensional structures, they are excellent models for understanding intramolecular force transmission as well as, more generally, the evolution of complex protein machines. The central goal of this proposal is to develop an understanding of the mechanisms and evolutionary divergence of such complex protein machines, leading to advances in our ability to predict and control the behaviors of cellular systems in both normal and disease states. The T4 bacteriophage (T4) is a small virus that infects the E. coli bacterium. The T4 genome encodes its own DNA replication proteins, including a sliding clamp and clamp loader, proteins that are closely related to their counterparts in eukaryotic cells, including human cells. We have developed and validated a powerful high- throughput functional assay for the T4 bacteriophage (T4) clamp loader system. This platform opens up many avenues to investigate mechanism and design principles in a proper biological context. We will use high- throughput mutagenesis to map mutational sensitivity and allosteric coupling in the clamp loader and examine the conservation of these properties in a very divergent AAA+ ATPase, a protein that controls transcription in bacteria. We will use statistical models trained on genome sequences to infer the essential constraints on and between amino acids in clamp loaders and test these inferences in a biological context. The aims of this project represent a unified body of work to use new assay systems to understanding clamp loader and AAA+ mechanism, and to test the potential of emerging sequence-based models for understanding and engineering complex macromolecular machines.