| The exquisite control and monitoring capability of single molecules has had an enormous impact on biology, particularly through the ability to monitor individual reactions at high temporal and spatial resolution. Not only have the physical properties of long polymeric molecules (e.g. DNA, microtubules) been elucidated, but real-time monitoring of individual molecular motors with which they interact has been achieved. Nonetheless, a fundamental limitation to most of these experiments is that they do not probe enzymatic behavior within its natural context of the living cell, but rather inside artificial chambers containing only a handful of purified components. The contrast with the cellular environment is large: the cell includes all components necessary for full activity of any biological process, but it also regulates the temporal sequence of biological processes and may present any given process with a very high local density of background molecules that compete for the same resources. Thus, the functioning of a molecular motor - its rates, processivity, substrate binding times, even whether it is at any given moment ?on? or ?off? - is potentially quite different inside the living cell. The extension of single-molecule techniques to the cell thus presents a fascinating opportunity. Using single-molecule sensitivity acquired through the localization of fluorescently-labelled molecular motors on DNA substrates [1], we will study the dynamics of the essential cellular process of DNA replication. We will focus on the arrest of this process by monitoring the timing of arrest, the removal of roadblocks, and the consequences of failure. We will primarily employ bacteria as model systems. System throughput will be optimized through the use of photoactivatable fluorophores and microfluidics, and control experiments on motor kinetics will be performed via in vitro single-molecule experiments. In this manner we will probe the biophysics of molecular motors in their natural environment to advance our understanding of the living cell. |
