The past decade has witnessed an emergence of technologies from various disciplines that are providing unprecedented opportunities to formulate and answer new types of biological questions. In particular, single-molecule approaches are revolutionizing biological inquiries by providing previously unattainable data on elementary biological processes. 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 in vivo conditions, we need to study more-complex systems with many components. A large part of my group's efforts is focused on pushing the single-molecule techniques to the extreme (e.g., ultrahigh spatiotemporal resolution, multicolor fluorescence, vesicle encapsulation, fluorescence and force combination) to extend the reach of the methods dramatically. We are also using the state-of-the-art single-molecule techniques to study the molecules that are important in maintaining the stability of the genome and in preventing serious threats to human health.
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 my research. Helicases are found in all three kingdoms of life and are extremely numerous: 1–2 percent of eukaryotic genes are helicases. Several severe human genetic diseases have been linked to mutations in helicases. Our helicase research is focused on three topics: (1) the motor mechanism, (2) functional diversity, and (3) intracellular localization and functions.
To study the activity of helicases or other enzymes at the molecular level, we need versatile tools that can deliver high spatial and temporal resolution. One such tool, single-molecule fluorescence resonance energy transfer (sm-FRET), utilizes the interaction between two different fluorophores—a donor and an acceptor that are typically attached to different locations on a biological molecule—to measure the conformational changes during function.
The most fundamental activity of all helicases is translocation, the ability to move along nucleic acids powered by ATP hydrolysis; hence helicases are motor proteins. How this motor works remains a mystery. We are tackling this question by looking at movements of individual superfamily 1 helicases (Rep, UvrD, and PcrA) in real time (collaboration with Timothy Lohman [Washington University in St. Louis]). We measure sm-FRET between various sites on the protein and on the DNA to build dynamic structural models of the protein-DNA complex during translocation. These studies have also resulted in the surprising finding of repetitive shuttling of the Rep, UvrD, and PcrA helicases on single-stranded DNA (ssDNA), which may indicate a new biological function of these enzymes (see Movie 1). Besides this repetitive shuttling activity, we have also observed a repetitive unwinding activity in a superfamily 2 helicase, the NS3 helicase from hepatitis C virus (collaboration with Anna Marie Pyle [HHMI, Yale University]). This study also provided the first functional evidence for the unwinding of a single base pair per ATP hydrolyzed and suggested a spring-loaded mechanism in which the enzyme accumulates tension on the DNA-protein complex up to about 3 bases translocation, which is periodically released to unwind about 3 base pairs in a burst (see Movie 2).
The structural information we obtain from FRET experiments also provides the benchmarks for all-atom molecular dynamics simulations (collaboration with Klaus Schulten [University of Illinois at Urbana-Champaign]). In a recent study, we showed that the unidirectional translocation of PcrA helicase on ssDNA is regulated allosterically through synchronization of ATP hydrolysis and domain mobilities. We are also aiming to engineer a helicase that reverses its natural direction. (This work is supported by the National Institutes of Health.)
We have initiated studies on human RecQ helicases that are mutated in Bloom syndrome and Werner syndrome. These helicases process a number of novel DNA motifs—such as telomeric G-quadruplex, Holliday junction, and stalled replication fork. How they achieve such diverse functions while utilizing the fundamental engine for DNA translocation is a fascinating question. Crucial to these studies are the structural dynamics of these novel DNA motifs themselves, and we have made progress in understanding the spontaneous dynamics of the Holliday junction (collaboration with David Lilley [University of Dundee, U.K.]) and the G-quadruplex. For example, our spontaneous branch migration studies with single–base pair resolution revealed a surprisingly rugged energy landscape of Holliday junction branch migration. We also extended our branch migration studies to the enzymatic process catalyzed by a ring-shaped T7 helicase (collaboration with Smita Patel [University of Medicine and Dentistry of New Jersey]). The data on this study suggest that the enzyme works as a Brownian ratchet by biasing otherwise random spontaneous branch migration.
The formation of a helical filament around DNA by RecA is a key step in homologous recombination, but the details of this highly dynamic process have remained enigmatic. We developed a single-molecule fluorescence and hidden Markov modeling approach that can follow the entire life cycle of the RecA filament with single-monomer resolution and milliseconds time resolution. About five RecA monomers are needed to nucleate the filament, which grows and shrinks at both ends, one monomer at a time. The dynamic interplay between the RecA filament and the single-stranded DNA-binding (SSB) protein could also be clearly elucidated. We are now studying the mechanism of homology search and strand exchange by the RecA filament. (This work is supported by the National Science Foundation.)
SSB protein is another system that serves a crucial role in maintaining the unwound single strands of the DNA open during cellular processes such as DNA replication or repair. We used sm-FRET to study the two main SSB-binding modes and their interconversion rate as a function of monovalent and divalent ion concentration of the media (collaboration with Timothy Lohman). Beyond these fundamental binding modes, we also observed diffusion of SSB on DNA, which could have important implications for the function of this protein.
Another main branch of my research is the study of biological processes on vesicle-encapsulated single molecules (see Figure 1). Compared to surface immobilization, this unconventional method provides some advantages and new controls. The obvious advantage is complete isolation of the molecule of study from the surface. In addition, effective concentration of one or a few molecules inside a vesicle is in the micromolar range, much larger than other single-molecule essays that are typically performed at picomolar concentration. Such high effective concentrations enable single-molecule fluorescence studies of very weak interactions. We also use porous vesicles so that the reaction buffer can be quickly exchanged while keeping the biomolecule inside the vesicle. We have used our expertise in this area to study the membrane fusion induced by SNARE proteins at the single-vesicle level (collaboration with Yeon-Kyun Shin [Iowa State University]). (This work is supported by the National Institutes of Health.)
We are also developing next-generation hybrid microscopes that combine single-molecule imaging with single-molecule manipulation. As a first step, we have built an apparatus combining sm-FRET with optical tweezers and measured force dependence of conformational changes in a Holliday junction (see Figure 2). This study generated a two-dimensional reaction landscape of a Holliday junction and provided a detailed structure of the transient states populated by the Holliday junction during its conformational changes. (This work is supported by the National Science Foundation.)
In addition to measuring distances, via FRET, and applying forces, via optical tweezers, we have used localization of single fluorophores to study single molecules. We have developed a technique that enabled us to localize a fluorophore with 1-nm precision and subsecond time resolution, leading to proof that myosin V, a cellular motor protein in charge of cargo transport, walks hand-over-hand (collaboration with Paul Selvin [University of Illinois at Urbana-Champaign] and Yale Goldman [University of Pennsylvania]). We extended this technique to measure two identical fluorophores with 10-nm resolution by utilizing digital photobleaching of a single fluorophore. We also extended our two-color FRET capabilities to three-color FRET, which allows us to measure more than one distance simultaneously. Another technical development that was realized in our lab is the suppression of quantum dot blinking under physiologically relevant conditions, which we are hoping to use to perform challenging single-molecule experiments within live cells.
