Single-molecule Measurements of DNA Topology and Topoisomerases

Topological properties of DNA influence its mechanical and biochemical interactions. Genomic DNA is maintained in a state of topological homeostasis by topoisomerases and is subjected to mechanical stress arising from replication and segregation. Despite their fundamental roles, the effects of topology and force have been difficult to ascertain. Developments in single-molecule manipulation techniques have enabled precise control and measurement of the topology of individual DNA molecules under tension. This minireview provides an overview of these single-molecule techniques and illustrates their unique capabilities through a number of specific examples of single-molecule measurements of DNA topology and topoisomerase activity.     

See me, feel me: methods to concurrently visualize and manipulate single DNA molecules and associated proteins

Direct visualization of DNA and proteins allows researchers to investigate DNA-protein interactions with great detail. Much progress has been made in this area as a result of increasingly sensitive single-molecule fluorescence techniques. At the same time, methods that control the conformation of DNA molecules have been improving constantly. The combination of both techniques has appealed to researchers ever since single-molecule measurements have become possible and indeed first implementations of such combined approaches have proven useful in the study of several DNA-binding proteins in real time. Here, we describe the technical state-of-the-art of various integrated manipulation-and-visualization methods. We first discuss methods that allow only little control over the DNA conformation, such as DNA combing. We then describe DNA flow-stretching approaches that allow more control, and end with the full control on position and extension obtained by manipulating DNA with optical tweezers. The advantages and limitations of the various techniques are discussed, as well as several examples of applications to biophysical or biochemical questions. We conclude with an outlook describing potential future technical developments in combining fluorescence microscopy with DNA micromanipulation technology.     

Single-molecule analysis of DNA replication in Xenopus egg extracts

The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.     

Single-Molecule Methods for Investigating the Double-Stranded DNA Bendability

The various DNA-protein interactions associated with the expression of genetic information involve double-stranded DNA (dsDNA) bending. Due to the importance of the formation of the dsDNA bending structure, dsDNA bending properties have long been investigated in the biophysics field. Conventionally, DNA bendability is characterized by innate averaging data from bulk experiments. The advent of single-molecule methods, such as atomic force microscopy, optical and magnetic tweezers, tethered particle motion, and single-molecule fluorescence resonance energy transfer measurement, has provided valuable tools to investigate not only the static structures but also the dynamic properties of bent dsDNA. Here, we reviewed the single-molecule methods that have been used for investigating dsDNA bendability and new findings related to dsDNA bending. Single-molecule approaches are promising tools for revealing the unknown properties of dsDNA related to its bending, particularly in cells.     

Characterisation of bacterial polysaccharides: steps towards single-molecular studies

Techniques used in studies of polysaccharides, including chemical composition, linkage pattern, and higher order structures are in constant development. They provide information necessary for understanding of the polysaccharide properties and functions. Here, recent advancements in studies of the polysaccharides at the single-molecule level are highlighted. Over the last few years, single-molecule techniques such as force spectroscopy have improved in sensitivity and can today be used to detect forces in the pN range. In addition, these techniques can be used to investigate properties of single molecules close to physiological conditions. The challenges in the interpretation of the observations are aided by control experiments using well-characterised polysaccharides and by data provided by complementary methods. This field is expected to have increasing impact on the further advancement of the molecular understanding of the role of polysaccharides in various biological processes such as recognition and cell adhesion.     

Single-molecule perspectives on helicase mechanisms and functions

Helicases are a diverse group of molecular motors that utilize energy derived from the hydrolysis of nucleoside triphosphates (NTPs) to unwind and translocate along nucleic acids. These enzymes play critical roles in nearly all aspects of nucleic acid metabolism, and consequently, a detailed understanding of helicase mechanisms at the molecular level is essential. Over the past few decades, single-molecule techniques, such as optical tweezers, magnetic tweezers, laminar flow, fluorescence resonance energy transfer (FRET), and DNA curtains, have proved to be powerful tools to investigate the functional properties of both DNA and RNA helicases. These approaches allow researchers to manipulate single helicase molecules, perturb their free energy landscape to probe the chemo-mechanical activities of these motors, and to detect the conformational changes of helicases during unwinding. Furthermore, these techniques also provide the capability to distinguish helicase heterogeneity and monitor helicase motion at nanometer spatial and millisecond temporal resolutions, ultimately providing new insights into the mechanisms that could not be resolved by ensemble assays. This review outlines the single-molecule techniques that have been utilized for measurements of helicase activities and discusses helicase mechanisms with a focus on functional and mechanistic insights revealed through single-molecule investigations in the past five years.     

Nanopore-based Fourth-generation DNA Sequencing Technology

Nanopore-based sequencers, as the fourth-generation DNA sequencing technology, have the potential to quickly and reliably sequence the entire human genome for less than $1000, and possibly for even less than $100. The single-molecule techniques used by this technology allow us to further study the interaction between DNA and protein, as well as between protein and protein.Nanopore analysis opens a new door to molecular biology investigation at the single-molecule scale.In this article, we have reviewed academic achievements in nanopore technology from the past as well as the latest advances, including both biological and solid-state nanopores, and discussed their recent and potential applications.     

Unraveling secrets of telomeres: One molecule at a time

Telomeres play important roles in maintaining the stability of linear chromosomes. Telomere maintenance involves dynamic actions of multiple proteins interacting with long repetitive sequences and complex dynamic DNA structures, such as G-quadruplexes, T-loops and t-circles. Given the heterogeneity and complexity of telomeres, single-molecule approaches are essential to fully understand the structure–function relationships that govern telomere maintenance. In this review, we present a brief overview of the principles of single-molecule imaging and manipulation techniques. We then highlight results obtained from applying these single-molecule techniques for studying structure, dynamics and functions of G-quadruplexes, telomerase, and shelterin proteins.     

Single-molecule studies of DNA replisome function

Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the leading and lagging strand synthesis of DNA. These complex, yet elegantly organized, molecular machines have been studied extensively by kinetic and structural methods to provide an in-depth understanding of the mechanism of DNA replication. Owing to averaging of observables, unique dynamic information of the biochemical pathways and reactions is concealed in conventional ensemble methods. However, recent advances in the rapidly expanding field of single-molecule analyses to study single biomolecules offer opportunities to probe and understand the dynamic processes involved in large biomolecular complexes such as replisomes. This review will focus on the recent developments in the biochemistry and biophysics of DNA replication employing single-molecule techniques and the insights provided by these methods towards a better understanding of the intricate mechanisms of DNA replication.     

Honey, I shrunk the DNA: DNA length as a probe for nucleic-acid enzyme activity

The replication, recombination, and repair of DNA are processes essential for the maintenance of genomic information and require the activity of numerous enzymes that catalyze the polymerization or digestion of DNA. This review will discuss how differences in elastic properties between single- and double-stranded DNA can be used as a probe to study the dynamics of these enzymes at the single-molecule level.     

Dissecting elastic heterogeneity along DNA molecules coated partly with Rad51 using concurrent fluorescence microscopy and optical tweezers

Nucleoprotein filament formation by recombinases is central to homologous recombination. To follow this process, we used fluorescent human Rad51 recombinase to visualize the interactions with double-stranded DNA (dsDNA). Fluorescence imaging revealed that Rad51 filament formation on dsDNA initiates from multiple nucleation points, resulting in Rad51-dsDNA nucleoprotein filaments interspersed with regions of bare DNA. The elastic properties of such heterogeneously coated DNA molecules were assessed by combining force-extension measurements using optical traps with fluorescence microscopy. This combination of single-molecule techniques allows discrimination of segments within an individual DNA molecule and determination of their elastic properties. The nonfluorescent zones of DNA-Rad51 constructs showed the well-known (over)stretching behavior of bare DNA. In contrast, the fluorescent, Rad51-coated zones did not overstretch and Rad51 remained stably bound in a structure that was approximately 50% longer than bare DNA. These results illustrate the power of adding sensitive fluorescence imaging to optical tweezers instrumentation.     

Magnetic tweezers: a sensitive tool to study DNA and chromatin at the single-molecule level.

The advent of single-molecule biology has allowed unprecedented insight into the dynamic behavior of biological macromolecules and their complexes. Unexpected properties, masked by the asynchronous behavior of myriads of molecules in bulk experiments, can be revealed; equally importantly, individual members of a molecular population often exhibit distinct features in their properties. Finally, the single-molecule approaches allow us to study the behavior of biological macromolecules under applied tension or torsion; understanding the mechanical properties of these molecules helps us understand how they function in the cell. In this review, we summarize the application of magnetic tweezers (MT) to the study of DNA behavior at the single-molecule level. MT can be conveniently used to stretch DNA and introduce controlled levels of superhelicity into the molecule and to follow to a high definition the action of different types of topoisomerases. Its potential for chromatin studies is also enormous, and we will briefly present our first chromatin results.     

Chromatin fibers,one-at-a-time

Eukaryotic DNA is presented to the enzymatic machineries that use DNA as a template in the form of chromatin fibers. At the first level of organization, DNA is wrapped around histone octamers to form nucleosomal particles that are connected with stretches of linker DNA; this beads-on-a-string structure folds further to reach a very compact state in the nucleus. Chromatin structure is in constant flux, changing dynamically to accommodate the needs of the cell to replicate, transcribe, and repair the DNA, and to regulate all these processes in time and space. The more conventional biochemical and biophysical techniques used to study chromatin structure and dynamics have been recently complemented by an array of single-molecule approaches, in which chromatin fibers are investigated one-at-a-time. Here we describe single-molecule efforts to see nucleosomes, touch them, put them together, and then take them apart, one-at-a-time. The beginning is exciting and promising, but much more effort will be needed to take advantage of the huge potential that the new physics-based techniques offer.     

DNA and chromatin imaging with super-resolution fluorescence microscopy based on single-molecule localization

With the expansion of super-resolution fluorescence microscopy methods, it is now possible to access the organization of cells and materials at the nanoscale by optical means. This review discusses recent progress in super-resolution imaging of isolated and cell DNA using single-molecule localization methods. A high labeling density of photoswitchable fluorophores is crucial for these techniques, which can be provided by sequence independent DNA stains in which photoblinking reactions can be induced. In particular, unsymmetrical cyanine intercalating dyes in combination with special buffers can be used to image isolated DNA with a spatial resolution of 30-40 nm. For super-resolution imaging of chromatin, cell permeant cyanine dyes that bind the minor groove of DNA have the potential to become a useful alternative to the labeling of histones and other DNA-associated proteins. Other recent developments that are interesting in this context such as high density labeling methods or new DNA probes with photoswitching functionalities are also surveyed. Progress in labeling, optics, and single-molecule localization algorithms is being rapid, and it is likely to provide real insight into DNA structuring in cells and materials.     

Using Single-Molecule Approaches to Understand the Molecular Mechanisms of Heat-Shock Protein Chaperone Function

The heat-shock proteins (Hsp) are a family of molecular chaperones, which collectively form a network that is critical for the maintenance of protein homeostasis. Traditional ensemble-based measurements have provided a wealth of knowledge on the function of individual Hsps and the Hsp network; however, such techniques are limited in their ability to resolve the heterogeneous, dynamic and transient interactions that molecular chaperones make with their client proteins. Single-molecule techniques have emerged as a powerful tool to study dynamic biological systems, as they enable rare and transient populations to be identified that would usually be masked in ensemble measurements. Thus, single-molecule techniques are particularly amenable for the study of Hsps and have begun to be used to reveal novel mechanistic details of their function. In this review, we discuss the current understanding of the chaperone action of Hsps and how gaps in the field can be addressed using single-molecule methods. Specifically, this review focuses on the ATP-independent small Hsps and the broader Hsp network and describes how these dynamic systems are amenable to single-molecule techniques.