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

With this award, Dr. John Chaput from the University of California, Irvine will investigate the mechanism of DNA synthesis using advanced X-ray crystallography tools. DNA is the blueprint that directs the molecular basis of life on our planet.

However, there remains an incomplete understanding of how polymerases (the enzymes responsible for DNA synthesis) make new copies of DNA in cells. We will study this problem using a method that will produce snapshot images of the enzyme with the atomic-level. The collection of images will be assembled to produce the equivalent of an animated movie showing the precise order of each step in the reaction pathway.

In addition to fundamental knowledge, detailed insights into the mechanism of DNA synthesis could lead to the design of new polymerases for applications in biotechnology. 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 crystallography provides a powerful method for studying the mechanism of enzymes by capturing intermediates that cannot be observed in their lowest energy state. Extending this concept to replicative DNA polymerases is a challenging problem as it requires synchronizing all of the enzymes inside a crystal so that each enzyme is catalyzing the same chemical reaction at the same time.

The process is further complicated by large conformational changes that can obscure intermediates that are short lived, but essential to DNA synthesis. While the order of intermediates in the pathway of a replicative DNA polymerase has been observed, further details about the chemical bond forming step and generality of the reaction cycle remain unknown.

The intellectual merit of this proposal is to close this gap by collecting time-lapsed images of the chemical bond forming step with complementary and non-complementary bases and validating the reaction cycle in a second DNA polymerase I member. The full collection of snapshot images will provide unequivocal evidence of the precise order of each intermediate in the reaction pathway and may lead to design principles that could be used to generate specialty polymerases for biotechnology.

The successful completion of this project is expected to illuminate the chemistry of DNA synthesis with atomic-level resolution.

This project is jointly funded by the Chemistry of Life Processes Program of the Division of Chemistry (CHE) in the Directorate for Mathematical and Physical Sciences (MPS) and by the Genetic Mechanisms Program of the Division of Molecular and Cellular Biosciences (MCB) in the Directorate for Biological Sciences (BIO).

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.