Summary of Research in Progress Since arriving at the NIH at the end of February 2007, I have been concerned principally with setting up the lab and ordering parts for the single molecule instrumentation we are building. In addition to the ongoing process of designing and building single-molecule optical and magnetic manipulation instruments, we are pursing two projects. The first project concerns the ability of type II topoisomerases to relax the topology of DNA to below equilibrium values. In vivo these topoisomerases are responsible for unlinking replicated chromosomes prior to cell division. Since even a single link between sister chromosomes can prevent division and induce cell death, it is important that these enzymes preferentially unlink rather than link DNA molecules. In vitro it was shown that indeed these enzymes preferentially unlink rather than link DNA. However the mechanism by which an enzyme that acts locally on the scale of nanometers can determine the global linking topology of micron sized DNA molecules remains a mystery. One proposed mechanism suggests that unlinking may be favored over linking if the topoisomerase induces a sharp bend in the DNA on binding. We are currently using atomic force microscopy to image type II topoisomerases bound to DNA. From these measurements we hope to extract the induced bend angle, which in combination with Monte Carlo simulations of DNA molecules with a given bend angle, will allow us to determine if the topoisomerase induced bending model can explain the observed unlinking/linking asymmetry. The second project concerns the longstanding problem of optically induced damage in optical trapping experiments. Optical traps provide a means of non-invasively manipulating micron sized objects with light. The objects that can be manipulated include single bacterial or spermatozoa, the organelles of larger cells or organisms, and micro-spheres used as handles to manipulate single molecules of DNA or single proteins. Optical traps can be configured to apply controlled forces ranging from one to over a hundred piconewtons and to measure displacements on the order of one nanometer or less. One limiting drawback of optical trapping experiments is that the sample is damaged by the laser light used to produce the trap. Whereas optically induced damage occurs in all optical trapping experiments, it is of particular concern for in vivo measurements. The precise origin of optically induced damage has not been identified, however the most likely candidate is singlet oxygen generated by the trapping laser light. To test this hypothesis and to explore the possibility of eliminating optically induced damage in optical traps, we are testing the damage induced by long wavelength trapping light. The rational is that by trapping with laser light that does not have enough energy to excite molecular oxygen to the singlet state, optically induced damage will be dramatically reduced or eliminated. We have chosen Escherichia coli (E. coli) cells as a model system in which to quantify optically induced damage. Single E. coli cells are held in the optical trap and their flagellar rotation frequency is determined from the scattered laser light. The accumulation of optically induced damage leads to a decrease in the flagellar rotation frequency. We will test the relative rate of damage at the most commonly used wavelength (1064 nm) and at a wavelength corresponding to an energy less than that necessary to excite single oxygen (>1300 nm). Future Research Plans In addition to completing the above projects, our immediate goal is to finish the design and construction of an optical trap, a magnetic tweezers and a single-molecule fluorescence instrument. These instruments will be employed to pursue the longer term goals of the lab. Initially we will focus on the interaction of type I topoisomerases with helicases. Helicases of the RecQ family and topoisomerase III have been shown to functionally and physically interact in organisms ranging from bacteria to humans. Disruption of the interaction between the two enzymes leads to severe chromosome instability however, the mechanisms underlying their interaction, and the specific activity of the coupled enzyme remain unclear. Analysis of the coupled enzyme system is complicated by the fact that both the helicase and the topoisomerase individually modify the structure of DNA, and these activities must be distinguished from the activity of the coupled enzymes. The ability of single-molecule techniques to measure the activity of a single enzyme or enzyme complex in real time is well suited to the study of such complicated processes in which multiple activities may occur over multiple time scales. Following the activity of a single enzyme or multi-enzyme complex over time can reveal transient phenomena, fluctuations in activity, and the presence of enzyme sub-populations or enzymatic states, all of which are obscured by the averaging inherent in traditional ensemble measurements. Initially, we will use two complementary approaches to investigate different aspects of the interactions between helicases and topoisomerases. In one project, we will investigate the activity of reverse gyrase, a unique topoisomerase from hyperthermophilic bacteria that is comprised of a helicase and a topoisomerase on a single polypeptide. Reverse gyrase serves as a model system in which to study the interaction of a helicase and a topoisomerase. Through the concerted activity of the two domains, reverse gyrase promotes the positive supercoiling (over winding) of DNA, however the mechanism underlying this activity remains speculative. Single-molecule experiments will allow us to probe the details of the supercoiling reaction, and in conjunction with non-hydrolysable ATP analogs and point mutations, will allow us to determine the molecular basis for communication between the helicase and topoisomerase domains. In the second project will use single-molecule fluorescence techniques, primarily fluorescence resonance energy transfer (FRET), to measure the binding kinetics of RecQ helicase and Topo III form E. coli in isolation and in the presence of a variety of DNA substrates and nucleotide cofactors. These experiments and the experimental techniques employed will pave the way for more complex experiments in which the activity and the association state of single enzymes and complexes will be assayed simultaneously using a combination of single molecule manipulation and single molecule visualization techniques.