Single-Molecule Enzymology: Fluorescence-Based and High-Throughput Methods

Graduate of Peter the Great St. Petersburg Polytechnic University, Russia (1996 MS diploma with honors (equivalent of cum laude) in physics/biophysics) and Osaka University, Japan (2000 PhD in biological sciences), Dr. Maria Spies is an Associate Professor of Biochemistry at the University of Iowa Carver College of Medicine. Spies’ research career has been focused on deciphering the intricate choreography of the molecular machines orchestrating the central steps in the homology directed DNA repair. Her doctoral research supported by the Japanese Government (MONBUSHO) Graduate Scholarship provided the first detailed biochemical characterization of archaeal recombinase RadA. In her postdoctoral work with Dr. Steve Kowalczykowski (UC Davis) supported by the American Cancer Society, Spies reconstituted at the single-molecule level the initial steps of bacterial recombination and helped to explain how this process is regulated. Spies’ laboratory at the University of Iowa emphasizes the molecular machinery of homologous recombination, how it is integrated into DNA replication, repair and recombination (the 3Rs of genome stability), and how it is misappropriated in the molecular pathways that process stalled DNA replication events and DNA breaks through highly mutagenic, genome destabilizing mechanisms. Her goal is to understand, reconstitute and manipulate an elaborate network of DNA recombination, replication and repair, and to harness this understanding for anticancer drug discovery. The Spies lab utilizes a broad spectrum of techniques from biochemical reconstitutions of the key biochemical reactions in DNA recombination, repair and replication, to structural and single-molecule analyses of the proteins and enzymes coordinating these reactions, to combined HTS/CADD campaigns targeting human DNA repair proteins. Work in Spies Lab has been funded by the American Cancer Society (ACS), Howard Hughes Medical Institute (HHMI), and is currently supported by the National Institutes of Health (NIH). She received several prestigious awards including HHMI Early Career Scientist Award and Margaret Oakley Dayhoff Award in Biophysics. She serves on the editorial board of the Journal of Biological Chemistry, and as an academic editor of the journal Plos-ONE. She is a permanent member and a chair of the American Cancer Society “DNA mechanisms in cancer” review panel. Associate Professor of Physics and Biophysics, Department of Physics, University of Illinois at Urbana-Champaign, USA

1. Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases T.L. Pavankumar, J.C. Exell and S.C. Kowalczykowski 2. Single Molecule Imaging with one Color Fluorescence Y. Qiu and S. Myong 3. Measuring Membrane Protein Dimerization Equilibrium in Lipid Bilayers by Single-Molecule Fluorescence Microscopy R. Chadda and J.L. Robertson 4. Fluorescent Labeling of Proteins in Whole Cell Extracts for Single-Molecule Imaging S.R. Hansen, M.L. Rodgers and A.A. Hoskins 5. Quantifying the Assembly of Multicomponent Molecular Machines by Single-Molecule Total Internal Reflection Fluorescence Microscopy E.M. Boehm, S. Subramanyam, M. Ghoneim, M.T. Washington and M. Spies 6. How to Measure Separations and Angles Between Intra-Molecular Fluorescent Markers K.I. Mortensen, J. Sung, J.A. Spudich and H. Flyvbjerg 7. Precisely and Accurately Inferring Single-Molecule Rate Constants C.D. Kinz-Thompson, N.A. Bailey, R.L. Gonzalez 8. Quantification of Functional Dynamics of Membrane Proteins Reconstituted in Nanodiscs Membranes by Single Turnover Functional Readout M.E. Moses, P. Hedegård and N.S. Hatzakis 9. Putting Humpty-Dumpty Together: Clustering the Functional Dynamics of Single Biomolecular Machines Such as the Spliceosome C.E. Rohlman, M.R. Blanco and N.G. Walter 10. Single Molecule FRET to Measure Conformational Dynamics of DNA Mimatch Repair Proteins J.W. Gauer, S. LeBlanc, P. Hao, R. Qiu, B. Case, M. Sakato, M.M. Hingorani, D.A. Erie and K.R. Weninger 11. Single Molecule Confocal FRET Microscopy to Dissect Conformational Changes in the Catalytic Cycle of DNA Topoisomerases S. Hartmann, D. Weidlich and D. Klostermeier 12. Probing the Conformational Landscape of DNA Polymerases Using Diffusion-Based Single-Molecule FRET J. Hohlbein and A.N. Kapanidis 13. Methods for Investigating DNA Accessibility with Single Nucleosomes M.D. Gibson, M. Brehove, Y. Luo, J. North and M.G. Poirier 14. Single-Molecule Fluorescence Studies of Fast Protein Folding Z. Wang, L.A. Campos and V. Muñoz 15. Single-Molecule Multi-Color FRET Assay for Studying Structural Dynamics of Biomolecules S. Lee, Y. Jang, S.-J. Lee and S. Hohng 16. A Multi-Color Single Molecule FRET Approach to Study Protein Dynamics and Interactions Simultaneously M. Götz, P. Wortmann, S. Schmid and T. Hugel 17. Interferometric Scattering Microscopy for the Study of Molecular Motors J. Andrecka, Y. Takagi, K.J. Mickolajczyk, L.G. Lipert, J.R. Sellers, W.O. Hancock, Y.E. Goldman and P. Kukura 18. Enzyme Kinetics in Femtoliter Arrays P. Mogalisetti and D.R. Walt