Visualizing protein–DNA interactions at the single-molecule level

Abstract

SummaryRecent advancements in single-molecule methods have allowed researchers to directly observe proteins acting on their DNA targets in real-time. Single-molecule imaging of protein-DNA interactions permits detection of the dynamic behavior of individual complexes that otherwise would be obscured in ensemble experiments. The kinetics of these processes can be monitored directly, permitting identification of unique sub-populations or novel reaction intermediates. Innovative techniques have been developed to isolate and manipulate individual DNA or protein molecules, and to visualize their interactions. The actions of proteins that have been visualized include: duplex DNA unwinding, DNA degradation, DNA packaging, translocation on DNA, sliding, superhelical twisting, and DNA bending, extension, and condensation. These single-molecule studies have provided new insights into nearly all aspects of DNA metabolism. Here we focus primarily on recent advances in fluorescence imaging and mechanical detection of individual protein-DNA complexes, with emphasis on selected proteins involved in DNA recombination: DNA helicases, DNA translocases, and DNA strand exchange proteins.

Single-molecule approaches to characterizing kinetics of biomolecular interactions

“…The ability to observe biochemical reactions at the level of a single molecule has greatly contributed to our understanding of the molecular mechanisms that define life [1][2][3][4][5][6][7][8][9][10][11]. A major strength of studying processes at the level of individual molecules lies in the direct measurement of distributions of molecular properties, rather than their ensemble averages.…”

On the influence of protein-DNA register during homologous recombination

“…[10][11][12][13][14][15] We have developed the single-molecule DNA curtains method to establish a more detailed understanding of the molecular mechanisms that contribute to homologous DNA recombination. [16][17][18][19] For studies of recombination, we use ssDNA curtains, where long ssDNA molecules are tethered to a lipid bilayer on the surface of a microfluidic sample chamber and then organized into "curtains" using a series of strategically positioned barriers to lipid diffusion; details describing the preparation of DNA curtains can be found in references.…”

Direct Fluorescent Imaging of Translocation and Unwinding by Individual DNA Helicases

“…A complete understanding requires direct measurement of individual events that make up the distribution of behaviors that comprise the ensemble. In recent decades, we have used single-molecule imaging to visualize the movement and actions of DNA motor proteins (Amitani, Baskin, & Kowalczykowski, 2006; Bianco et al, 2001; Handa, Bianco, Baskin, & Kowalczykowski, 2005; Liu, Baskin, & Kowalczykowski, 2013; Nimonkar, Amitani, Baskin, & Kowalczykowski, 2007; Rad, Forget, Baskin, & Kowalczykowski, 2015; Spies, Amitani, Baskin, & Kowalczykowski, 2007; Spies et al, 2003; Amitani, Liu, Dombrowski, Baskin, & Kowalczykowski, 2010; Forget, Dombrowski, Amitani, & Kowalczykowski, 2013; Hilario & Kowalczykowski, 2010; Sun & Wang, 2016). Here, we limit ourselves to a description of some of those methods.…”

“…However, the study of protein–DNA interactions at the single-molecule level is supported by a wide range of complementary techniques, including, but not limited to, atomic force microscopy, electron microscopy, force spectroscopy, fluorescence correlation spectroscopy, Förster resonance energy transfer, magnetic tweezers, optical traps combined with fluorescence microscopy, and total internal reflection fluorescence (TIRF) microscopy (Bustamante, Bryant, & Smith, 2003; Greenleaf, Woodside, & Block, 2007; Ha, Kozlov, & Lohman, 2012; Kapanidis & Strick, 2009; Lionnet et al, 2012; Moffitt, Chemla, Smith, & Bustamante, 2008; Neuman & Nagy, 2008; Qi & Greene, 2016; Roy, Hohng, & Ha, 2008; Spies, 2013; Yodh, Schlierf, & Ha, 2010). Mechanical manipulation of individual DNA molecules by optical trapping of attached microspheres or by attachment to a surface has been a useful platform for studying the mechanisms of DNA helicases and translocases (Amitani et al, 2006, 2010; Bianco et al, 2001; Forget et al, 2013; Hilario & Kowalczykowski, 2010; Liu et al, 2013; Rad et al, 2015; Sun & Wang, 2016). …”