Elucidating the Hidden Steps of Replicative DNA Synthesis by Time-Resolved X-ray Crystallography

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With this award, the Chemistry of Life Processes program in the Chemistry Division and the Genetic Mechanisms program in the Division of Molecular and Cellular Biosciences are funding Dr. John Chaput from the University of California, Irvine to apply time-resolved X-ray crystallography methods to capture snapshot images as the DNA polymerase enzyme copies one strand of DNA onto another. DNA is the blueprint that directs the molecular basis of life on our planet. However, despite decades of research, there remains an incomplete understanding of how enzymes make new copies of DNA inside cells. The collection of images is assembled to produce the equivalent of an animated movie showing the precise order of each step in the reaction pathway at the atomic level. The detailed insights into the chemical mechanism of these enzymes leads potentially to the design of new polymerases for applications in biotechnology. In addition, this project includes a significant educational component that is designed to attract and maintain student interest in the chemical and biological sciences. This includes pro-active engagement in a number of university-sponsored programs, including the Minority Science Program, aimed at improving the participation of traditionally underrepresented groups in science. Time-resolved X-ray crystallography is a powerful method for studying the mechanism of enzymes by capturing intermediates that cannot be observed in the lowest energy states of static protein crystal structures. This project investigates the mechanism of DNA synthesis by collecting time-lapsed images of the Bacillus stearothermophilus (Bst) and the Klenow-fragment analogue of the Thermus aquaticus (KlenTaq) DNA polymerase. The objectives are to provide details of a tyrosine gating mechanism that prevents frame-shift mutations and a novel push-pull mechanism during elongation. In addition, the study probes an unusual alternative reaction catalyzed by Bst polymerase that uses non-canonical templates (RNA, 2-fluoro-arabino nucleic acid, or alpha-L-threose nucleic acid) for DNA elongation. The time-lapse structures are projected to identify conformational changes as they occur in the enzyme active site. The full collection of snapshot images is to provide unequivocal evidence for the precise order of each intermediate in the reaction pathway and may lead to the discovery of new intermediates not observed in previous DNA polymerase studies. Successful completion of this project is expected to illuminate the chemistry of one of life’s most essential processes—DNA synthesis.This award is co-funded by the Chemistry of Life Processes program in the Division of Chemistry (Mathematical and Physical Sciences (MPS) Directorate) and the Genetic Mechanisms cluster in the Division of Molecular and Cellular Biosciences (Biological Sciences (BIO)Directorate).This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.