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Single-Molecule Studies of Genomic Maintenance

Research Summary

Taekjip Ha uses sophisticated physical techniques to manipulate and visualize the movements of single molecules to understand basic biological processes involving DNA and other molecules.

Single-molecule detection has opened vast avenues to investigate aspects of biological systems that are inaccessible by any other technique. However, most of the single-molecule studies have been limited to isolated single protein, RNA, and DNA molecules, yet these molecules do not function in isolation in the cell. To better emulate the cellular environment, we need to study more-complex systems with many components.

Research in my lab is focused on pushing the limits of single-molecule detection methods to study biological systems as a complex. To do this, we develop state-of-the-art techniques (e.g., multicolor fluorescence, superresolution imaging, combined force and fluorescence spectroscopy, vesicular encapsulation, quantitative fluorescence labeling strategies). These techniques are enabling us to test diverse protein–nucleic acid and protein-protein complexes and the mechanistic basis of their interactions and functions – both in vitro and in vivo – at an unprecedented spatial and temporal resolution.

Conformational Dynamics of Nucleic Acids
The elastic model of DNA duplex dynamics predicts high bending stiffness in DNA polymers less than 150 base pairs (bp) in length. We recently developed a fluorescence resonance energy transfer (FRET)-based cyclization assay to explain the apparent bendability of DNA <100 bp observed in many cellular processes, such as gene expression regulation, packaging in viral capsids, and nucleosome formation. We can determine the effect of DNA length on the rates and variation of DNA looping. The DNA cyclization assay allows us to see the effect of various DNA modifications, defects, and proteins on DNA flexibility. We are also utilizing three-color FRET strategies and MD-Gō simulations (in collaboration with Sarah Woodson [Johns Hopkins University] and Zaida Luthey-Schulten [University of Illinois at Urbana-Champaign]) to understand the sequential events in ribosome assembly. Our studies of ribosomal protein S4 binding to Escherichia coli 16S rRNA have captured the cooperativity in simultaneous binding and folding events and shed more light on protein-RNA interactions during the ribosome assembly process.

Movie 1: A helicase protein moves rapidly on highly flexible single-stranded DNA track, using power provided by ATP hydrolysis. Once the helicase encounters a physical blockade that it cannot surmount, a conformational change in the helicase protein results in the recruitment of the initial site on the DNA, forming a loop. The helicase protein then snaps back to the beginning site on the DNA and repeats the movement. Repetitive movement on the DNA may keep it clear of potentially toxic proteins.

Animation: Courtesy of Taekjip Ha

Movie 2: The three domains of the hepatitis C helicase, NS3, are represented in blue, green, and yellow. The blue and green domains walk along the double-stranded DNA (ladder-like structure, in black), one base pair (rungs) at a time, likely consuming one ATP (in red) per base pair. The yellow domain lags behind. These small reactions build tension in the DNA-protein complex, which unwinds only after three nucleotide pairs are unhitched from one another. When the enzyme encounters an insurmountable barrier, it bounces back and restarts the unwinding process.

Animation: Courtesy of Sua Myong and Taekjip Ha

Single-Molecule Study of Nucleic Acid–Binding Proteins
Helicases – the DNA/RNA-unwinding enzymes – and their enzymatic activities are associated with virtually all cellular processes involving nucleic acids and constitute an important aspect of our research. Helicases are found in all three kingdoms of life and are extremely numerous: an estimated 1–2 percent of eukaryotic genes encode helicases. Several severe human genetic diseases have been linked to mutations in these proteins. Using our small-molecule FRET approaches, we have gained insights into the functions of different families of helicases (e.g., Rep, NS3, UvrD, PcrA) and their role during DNA replication (e.g., T7). For example, we discovered that PcrA helicase reels in DNA in single base steps, forming a DNA loop and at the same time efficiently removing other proteins bound to the DNA.

We also discovered that NS3 helicase from a human pathogen, hepatitis C virus, uses a spring-loaded mechanism to unwind DNA. Furthermore, we determined that a "priming loop" formed during double-stranded DNA unwinding by T7 helicase enables coordination of leading- and lagging-strand synthesis during DNA replication. With respect to other DNA-binding proteins, we have used three-color FRET strategy to show that the sliding mechanism of RecA filament is crucial for fast homology search during DNA repair and recombination.

To elucidate the functional behavior of DNA-binding proteins in more detail, we have recently developed a next generation of single-molecule instrumentation combining ultrahigh-resolution optical traps, capable of detecting motions at the subnanometer scale, and single-molecule fluorescence detection. The instrument uses a high-speed interlacing scheme to combine dual optical traps with a confocal fluorescence microscope. This instrument will enable us to make direct correlation, for the first time, between conformational states and unwinding steps of helicases. In addition, we have recently built an instrument combining total internal reflection fluorescence microscopy with optical tweezers (fleezers) so that we can track movements of fluorescently labeled molecules on a stretched single-stranded DNA (ssDNA). This platform will allow us to probe the consequences of an encounter between a helicase and other DNA-bound proteins, which are difficult to probe using FRET alone. Insights into mechanisms of single-stranded DNA-binding protein (SSB) sliding on ssDNA are an example. We are expanding fleezer technology to study nucleosome conformational dynamics, particularly with respect to DNA wrapping and unwrapping on the histone octamers.

Visualizing Protein-Protein and Protein–Nucleic Acid Interactions
While we are taking advantage of single-molecule techniques to study biological molecules in vitro, we are also interested in understanding biological processes in their physiological context. We have developed a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional immunoprecipitation assay with single-molecule fluorescence microscopy to enable direct visualization of individual cellular protein complexes from a variety of sources. We have utilized this method for diverse complex systems to detect rare subpopulations of protein complexes and determine their stoichiometric composition in their cellular environment (in collaborations with Peter Cresswell [HHMI, Yale School of Medicine], Supriya Prasanth [University of Illinois at Urbana-Champaign], and Jie Chen [UIUC]).

For visualization within a cell, we have constructed a two-color three-dimensional (3D) superresolution optical microscope. Because living cells house many nanometer-sized molecules that are densely packed into assemblies and networks, traditional light microscopes, with their low resolving power, are insufficient. Our superresolution fluorescence imaging achieves spatial resolution of 50 nm in all three dimensions; this allows us to spatially localize and rigorously investigate the target search kinetics of regulatory noncoding RNA.

Mechanical Perturbation and Response of Biological Systems
To study force-dependent cellular function, we are developing probes to target a variety of cellular receptors that allow us to sense forces either by FRET sensing or rupture sensing, wherein we observe rupture of a sensor calibrated to rupture at a predefined force. Our fleezers instrument has enabled FRET versus force calibration at a much greater force and distance sensitivity. We have demonstrated FRET-force sensing by inserting an elastic peptide between the TFP-Venus fluorescent protein FRET pair. By placing this sensor in between the Vt and Vh domains of the protein vinculin, we have been able to achieve quantification of real-time focal adhesion formation by vinculin in live cells (in collaboration with Martin Schwartz [University of Virginia], Christopher Chen [University of Pennsylvania], and Stephen Sligar [UIUC]). We are also interested in understanding molecular forces that regulate cell surface receptor activation on the single-cell level. We are applying rupture sensing to study long-term effects due to force intervention in cell–extracellular matrix (ECM) interaction. To understand the collective cell-adhesion process, we are using rupture sensors based on DNA to measure forces experienced by single integrin-RGD complexes for different cell types.

Grants from the National Institutes of Health and the National Science Foundation provided partial support for these projects.

As of April 7, 2016

Scientist Profile

Johns Hopkins University
Biophysics, Structural Biology