David Millar, PhD

Biophysical Chemistry

The focus of our research is on the dynamic structural changes that occur during cellular rearrangements of DNA and during ribozyme catalysis. One project is aimed at understanding the proofreading process that underlies the high DNA replication fidelity of DNA polymerases. Using time-resolved and single-molecule fluorescence methods, in conjunction with site-directed mutagenesis procedures, we are elucidating the molecular mechanisms that control the movement of a DNA substrate between the polymerization site of the enzyme and a separate 3'-5' exonuclease site used in proofreading. We use similar methods to monitor large-scale conformational changes that occur during the catalytic cycle of the hairpin ribozyme, a small endonucleolytic RNA that serves as a model for much larger catalytic RNA molecules. By means of fluorescence resonance energy transfer (FRET) between appropriately placed fluorescent probes, we follow the docking of the substrate-binding and catalytic domains of the ribozyme in real time and identify nucleotides that stabilize the catalytically active docked conformation. Single-molecule FRET methods are also being applied to Holliday junctions, four-stranded DNA structures that are formed during the course of genetic recombination and related DNA repair events.