Our long term goal is to understand the mechanisms of enzymes involved in DNA replication and how mutations in helicases lead to human diseases. Helicases are integral components of the replication machinery in which their motor function is required to unwind dsDNA and to recombine DNA molecules. Defects in the functions of helicases cause a variety of human diseases including cancer, premature ageing, and neuromuscular disorders. Phage T7 encodes a ring shaped helicase-primase (T7 gp4), which is highly homologous to the human mitochondrial DNA helicase, Twinkle. The T7 proteins are model proteins to understand the molecular basis for numerous diseases associated with mutations in mtDNA helicase Twinkle and mtDNA polymerase gamma. Our studies of T7 gp4 in the last funding period highlighted the importance of studying replication proteins in association with their interacting partners. In the proposed studies, we will study the helicase and primase activities of T7 gp4 in association with T7 DNA polymerase and T7 single strand binding protein. We will extend our characterization of T7 gp4 from the last funding period to address key questions that arose from prior results: How is helicase rate accelerated by T7 DNAP? Does DNAP increase the rate and the step size of the helicase (bp unwound per nucleotide hydrolyzed)? How is the primase function coordinated with the helicase and polymerase functions? When are primers made? How does the lagging strand polymerase, which synthesizes DNA discontinuously, keep up with the leading strand polymerase? Concomitantly, we propose to initiate studies of the mtDNA helicase, Twinkle, and use selected T7 gp4 mutants as model proteins to understand the disease causing mutants of Twinkle. Some of the uncoupled T7 gp4 mutants will serve as tools to identify the principles of mechanochemical coupling. The studies will be carried out with the following specific aims: (1) to investigate the physical and functional interactions between helicase and polymerase. (2) To investigate the kinetic coupling between leading and lagging strand DNA synthesis. (3) To study T7 gp4 mutants homologous to the disease causing mutants in mitochondrial helicase Twinkle and to initiate studies on Twinkle and mitochondrial DNA replication.
DNA replication is an essential process of life that when disrupted leads to genome instability and serious human health problems. Mutations in human helicases manifest in a variety of diseases including cancer, premature aging, and mtDNA related disorders that are a leading cause of neurological diseases. This study will use T7 proteins as a model system to understand the basis of some of the mitochondrial related diseases caused by mutations in the helicase Twinkle. Phage T7 replication has served as a paradigm to understand DNA replication being remarkably similar to the replication complex of the human mitochondria. Understanding the structure and mechanisms of the proposed helicases is important in finding the link between the complex human diseases and defects in helicases as well as to aid in the drug discovery process. Future applications of this basic research could also be in designing nanomachines based on helicase motor for transporting materials such as sensors in the cell.
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