Understanding the Librarians of the Genome: developing new physical assays to detect conformational changes in DNA-processing enzymes
DNA polymerases, enzymes responsible for replicating and maintaining DNA in all domains of life, have been extensively studied over the past half-century. Despite the scrutiny, much remains unknown about the mechanism by which polymerases catalyze the building of a DNA strand and what limits their rate of reaction. Fluorescence techniques, Förster Resonance Energy Transfer (FRET) in particular, have allowed analysis of enzyme structure by using dyes as 'molecular rulers' of macromolecules. However, these methods cannot observe the smaller changes below 30 Å that occur at the active sites of enzymes. This project will extend FRET to the scale needed to study catalytic mechanisms of enzymes through the use of transition metal cation complexes as FRET acceptors over 10-20 Å, and examine the changes that occur in the active site of DNA polymerases.
The proposed research and training plan lies at the intersection of biology (enzyme function, genetics) and physics (optics, photophysics). The applicant will gain training in the physical sciences, particularly on the theory and application of single-molecule FRET, the use of high-level optical instrumentation, and statistical data analysis, building upon the applicant's biology and genetics background. The extension of FRET to previously-inaccessible distances will find broad applicability to studying small structural changes in large molecules -- biological and otherwise. More broadly, this project facilitates broad dissemination of its results by integrating cutting-edge research with undergraduate education (course development, teaching and student mentoring) and public outreach via local partner organizations.
