Single-Molecule Studies of DNA Replication: The Plasticity of Multi-Protein Complexes

Saturday, February 11, 2017 protein adsorption at a glass surface, previously unseen dynamics are revealed. 35-Subg Bright and Stable External Fluorophores in Untransformed Living Cells Ozgur Sahin. Biological Sciences and Physics, Columbia University, New York, NY, USA. We will present atomic force microscopy methods for imaging mechanical properties of single bio molecules and live cells. Our analysis of these images have yielded a set of equations that relate nanoscale stiffness of cells to intracellular forces. 36-Subg Single-Molecule Studies of DNA Replication: The Plasticity of MultiProtein Complexes Antoine M. van Oijen. School of Chemistry, University of Wollongong, Wollongong NSW, Australia. Advances in optical imaging and molecular manipulation techniques have made it possible to observe individual enzymes and record molecular movies that provide new insight into their dynamics and reaction mechanisms. In a biological context, most of these enzymes function in concert with other enzymes in multi-protein complexes, so an important new direction is the utilization of single-molecule techniques to unravel the orchestration of large macromolecular assemblies. We are applying a single-molecule approach to study DNA replication, a process that is supported by a large, multi-protein complex containing a number of different activities and held together by protein-protein and protein-DNA interactions of many different strengths. While the classical textbook picture of the replisome is one in which the protein components, in particular DNA polymerases, are efficiently retained and re-used for a large number of cycles of Okazaki-fragment synthesis on the lagging strand, our single-molecule studies imply a very different picture. I will present fluorescence-based single-molecule imaging experiments, in vitro and in vivo, of the E. coli DNAreplication machinery and demonstrate that new DNA polymerases are recruited by the replisome on a timescale of mere seconds. Further, simultaneous observation of both leading- and lagging-strand synthesis in a single replisome suggests that the replication machinery employs a number of different molecular mechanisms to achieve coordination between the DNA polymerases on each strand. 37-Subg Single Molecule Fluorescence and Atomic Force Microscopy Studies of DNA Repair Dorothy Erie1, Kira Bradford1, Hunter Wilkins1, Jacqueline Bower1, Zimeng Wang1, Jacob Gauer1, Matthew Satusky1, Rouyi Qiu2, Keith Weninger2, Parminder Kaur2, Hong Wang2. 1 Dept. Chemistry, University North Carolina, Chapel Hill, Chapel Hill, NC, USA, 2Dept. Physics, North Carolina State University, Raleigh, NC, USA. DNA polymerases that are responsible for replication make approximately one error for every 107 bases copied, but the human genome contains ~6 billion bases, which results in ~600 errors per round of replication. The DNA mismatch repair (MMR) system corrects these DNA synthesis errors that occur during replication. MMR is initiated by the highly conserved MutS and MutL homologs, which are both dimers and contain DNA binding and ATPase activities that are essential for MMR in vivo. MutS homologs initiate repair by binding to a mismatch and undergoing an ATPdependent conformational change that promotes its interaction with MutL homologs. This complex signals the initiation of excision and resynthesis of the newly synthesized DNA strand containing the incorrect nucleotide. We have been using a combination of atomic force microscopy (AFM) and single molecule fluorescence to characterize the stoichiometries and the conformational and dynamic properties of MutS and MutL homologs and their assembly on DNA containing a mismatch. We have also developed a new dual resonance frequency enhanced electrostatic force microscopy (DREEM), in which we simultaneously collect the AFM topographic image and an image of the electrostatic potential of the surface. The DREEM images reveal the path of DNA inside individual protein-DNA complexes, yielding unprecedented details about DNA conformations within simple and complicated complexes. I will discuss our studies on the assembly of MutS and MutL homologs on mismatches, with a focus on how AFM, DREEM, and single-molecule fluorescence can be powerful tools to study the stoichiometries, conformations, and dynamic assembly of multicomponent complexes.

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38-Subg Developing Fluorescent Nanodiamonds for In Vitro and In Vivo Biological Imaging Keir C. Neuman. Laboratory of Single Molecule Biophysics, National Institutes of Health, Bethesda, MD, USA. Fluorescent nanodiamonds are biocompatible fluorescent particles with indefinite photo-stability that make them superior in vitro and in vivo imaging probes for a wide range of applications. Fluorescence arises from specific defect centers within the nanodiamond lattice, which permits the generation of fluorescent nanodiamonds with different emission wavelengths, or combinations of emission wavelengths. The negatively charged nitrogen-vacancy (NV ) center is a defect in the diamond lattice consisting of a substitutional nitrogen and a lattice vacancy that form a nearest-neighbor pair. NV centers are fluorescent sources with remarkable optical properties including quantum efficiency near unity, indefinite photo-stability, i.e., no photobleaching or blinking, broad excitation spectra, and exquisitely sensitive magnetic field-dependent fluorescence emission. In particular, their near infrared fluorescence makes them ideally suited for in vivo imaging. However, producing, functionalizing, and characterizing small bright FNDs for biomedical applications remains challenging. We have developed multiple biocompatible functionalization schemes that permit the stabilization and specific labeling of fluorescent nanodiamonds. I will illustrate the unique features and potential uses of FNDs including high resolution three dimensional single-molecule imaging, in vivo background-free imaging through magnetic modulation of FND emission, and as superior fiducial markers for super-resolution microscopies. 39-Subg Spatially Resolved Mapping of Endogenous Proteins and RNA in Living Cells Alice Yen Ping Ting. Stanford University Medical School, Stanford, CA, USA. I will describe our efforts to build molecular tools for studying mitochondria and the synapse in living cells and tissue. These might include tools for proteomic mapping, transcriptomic mapping, and/or neural circuit mapping.

Biological Fluorescence Subgroup 40-Subg RESOLFT Optical Nanoscopy for the Life Sciences Ilaria Testa. Science for Life Laboratory, KTH Royal Institute of Technology, Stockholm, Stockholm, Sweden. Lens-based microscopy was unable to discern fluorescently labeled features closer than 200 nm for decades, until the recent breaking of the diffraction resolution barrier by sequentially switching the fluorescence capability of adjacent features on and off quickly made nanoscale imaging routine. Reported nanoscopy variants switch these features either in a target manner with intense laser beams, or molecule by molecule followed by computation in a stochastic fashion. Here, we show that emergent RESOLFT fluorescence nanoscopy enables fast and continuous imaging of living cells and tissues in super resolved detail by producing raw data images using only ultralow levels of light. This advance has been facilitated by the generation of fluorescent proteins (rsFP) that can be reversibly photoswitched numerous times. Distributions of functional rsFP-fusion proteins in living bacteria and mammalian cells are imaged with a spatial resolution less than 50nm. Using a fastswitching rsFP variant, we increased the imaging speed 50-fold over our first reported RESOLFT schemes, which in turn enabled us to record spontaneous and stimulated changes of dendritic actin filaments and spine morphology occurring on time scales from seconds to hours. Furthermore, lifetime based experiments enable precise localization of synapses by simultaneous recording of proteins located in the pre and post synaptic side. Our powerful RESOLFT technique represents a new paradigm for non invasive induction and monitoring of ultrastructural dynamics of synaptic plasticity at the nanoscale. 41-Subg Bright and Stable External Fluorophores in Untransformed Living Cells Paul R. Selvin. Physics, University of Illinois at Urbana-Champaign, Urbana, IL, USA. We have come up with a technique, relying on the well-known bacterial protein Streptolysin O (SLO), where we can take fluorophores bound to