Single-molecule investigations of fast biomolecular search processes initiating DNA repair

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The overarching objective of my research program is to create a new mechanistic understanding of the fast molecular search processes, which underlie a host of key cellular processes. One class of fast molecular search processes, that remain largely uncharacterized due to limited analysis techniques, is the search that initiates the constant processes of DNA repair following its cleavage by ionizing radiation, free radicals and during replication. Existing microscopy techniques are not capable of characterizing these molecular searches under physiological conditions because they can not simultaneously resolve: weak protein-DNA and DNA-DNA interactions; extended search trajectories of individual molecules over several seconds, and millisecond-timescale interaction kinetics; nor are they compatible with topologically complex or strained DNA substrates. I will circumvent these limitations using a new suite of custom single-microscopy techniques which extend CLIC-imaging. My first short-term objective is to create a unique in vitro microscopy station, capable of addressing open questions about the molecular searches performed by the well-calibrated MRX repair protein complex, whose biophysical properties and DNA-interactions have been measured previously, though under a limited range of conditions. My second short-term objective is to apply these techniques to perform the first single-molecule investigations of the 9-1-1 repair protein complex, which has only been studied with biochemical, cell-biological and genetic assays. My long-term objective is to extend CLIC-imaging to create a unique force and fluorescence in vitro microscopy station, which I will apply to probe molecular searches on DNA substrates of controlled topology and applied force. This research program sets the stage for the first single-molecule investigations of a wide range of molecular search processes (e.g. transcription and translation of messenger RNA) under conditions that systematically approach the complex cellular milieu, with myriad applications to biomedical research.