Terence R. Strick

Estimating the Persistence Length of a Worm-Like Chain Molecule from Force-Extension Measurements

We describe a simple computation of the worm-like chain model and obtain the corresponding force-versus-extension curve. We propose an improvement to the Marko and Siggia interpolation formula of Bustamante et al (Science 1994, 265:1599-1600) that is useful for fitting experimental data. We apply it to the experimental elasticity curve of single DNA molecules. Finally, we present a tool to study the agreement between the worm-like chain model and experiments.

Behavior of Supercoiled DNA

We study DNA supercoiling in a quantitative fashion by micromanipulating single linear DNA molecules with a magnetic field gradient. By anchoring one end of the DNA to multiple sites on a magnetic bead and the other end to multiple sites on a glass surface, we were able to exert torsional control on the DNA. A rotating magnetic field was used to induce rotation of the magnetic bead, and reversibly over- and underwind the molecule. The magnetic field was also used to increase or decrease the stretching force exerted by the magnetic bead on the DNA. The molecules degree of supercoiling could therefore be quantitatively controlled and monitored, and tethered-particle motion analysis allowed us to measure the stretching force acting on the DNA. Experimental results indicate that this is a very powerful technique for measuring forces at the picoscale. We studied the effect of stretching forces ranging from 0.01 pN to 100 pN on supercoiled DNA (-0.1 < sigma < 0.2) in a variety of ionic conditions. Other effects, such as stretching-relaxing hysteresis and the braiding of two DNA molecules, are discussed.

Single-molecule analysis of DNA uncoiling by a type II topoisomerase

Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment. This enables them to untangle DNA and relax the interwound supercoils (plectonemes) that arise in twisted DNA. In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death. Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled. By monitoring the DNAs extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the clamping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.

Twisting and stretching single DNA molecules

The elastic properties of DNA are essential for its biological function. They control its bending and twisting as well as the induction of structural modifications in the molecule. These can affect its interaction with the cell machinery. The response of a single DNA molecule to a mechanical stress can be precisely determined in single-molecule experiments which give access to an accurate measurement of the elastic parameters of DNA.

Abortive initiation and productive initiation by RNA polymerase involve DNA scrunching

Using single-molecule DNA nanomanipulation, we show that abortive initiation involves DNA “scrunching”—in which RNA polymerase (RNAP) remains stationary and unwinds and pulls downstream DNA into itself—and that scrunching requires RNA synthesis and depends on RNA length. We show further that promoter escape involves scrunching, and that scrunching occurs in most or all instances of promoter escape. Our results support the existence of an obligatory stressed intermediate, with approximately one turn of additional DNA unwinding, in escape and are consistent with the proposal that stress in this intermediate provides the driving force to break RNAP-promoter and RNAP-initiation-factor interactions in escape.

Stretching of macromolecules and proteins

In this paper we review the biophysics revealed by stretching single biopolymers. During the last decade various techniques have emerged allowing micromanipulation of single molecules and simultaneous measurements of their elasticity. Using such techniques, it has been possible to investigate some of the interactions playing a role in biology. We shall first review the simplest case of a non-interacting polymer and then present the structural transitions in DNA, RNA and proteins that have been studied by single-molecule techniques. We shall explain how these techniques permit a new approach to the protein folding/unfolding transition.

Biology, one molecule at a time

Single-molecule techniques have moved from being a fascinating curiosity to a highlight of life science research. The single-molecule approach to biology offers distinct advantages over the conventional approach of taking bulk measurements; this additional information content usually comes at the cost of the additional complexity. Popular single-molecule methods include optical and magnetic tweezers, atomic force microscopy, tethered particle motion and single-molecule fluorescence spectroscopy; the complement of these methods offers a wide range of spatial and temporal capabilities. These approaches have been instrumental in addressing important biological questions in diverse areas such as protein-DNA interactions, protein folding and the function(s) of membrane proteins.

The Manipulation of Single Biomolecules

By monitoring the response of individual protein and DNA molecules to pulling and twisting, biophysicists can learn much about their structure and their interactions.

Single-molecule DNA nanomanipulation: improved resolution through use of shorter DNA fragments

Single-molecule nanomanipulation of supercoiled DNA permits measurement, in real time, of spatial and temporal parameters of protein-DNA interactions that affect DNA topology 1–7 . In this method, a double-stranded DNA molecule containing at least one target for the protein of interest is attached at one end to a magnetic bead and at the other end to a glass surface. The experimental setup and the monitoring of the end-to-end extension (l) of the stretched, supercoiled DNA molecule is diagramed in Figure 1a. The protein of interest is introduced into the system, and protein-dependent changes in DNA linking number (Lk) or DNA twist (Tw) are detected as changes in the number of plectonemic supercoils (changes in DNA writhe, Wr; Lk = Tw + Wr; ref. 8) and corresponding changes in l (Fig. 1b–d). This approach has been applied to analysis of supercoil formation and relaxation by topoisomerases 1–5 and to promoter unwinding by bacterial RNA polymerase (RNAP) 6,7 . The spatial and temporal resolution of the method is expected to increase with decreasing length of the supercoiled DNA segment (equations in ref. 6). (Reducing the length of the supercoilable DNA segment should have no effect on the amplitude of changes in DNA extension resulting from protein-dependent changes in DNA topology (signal), but should reduce the amplitude of random fluctuations in DNA extension (noise), thereby resulting in improvement in the signal-to-noise ratio.) Previous work has involved supercoiled DNA segments 4–44 kilobases (kb) in length 1–7 . Here, we describe preparation of DNA molecules with 2-kb supercoilable DNA segments and document superior resolution in analysis of promoter unwinding and DNA compaction by bacterial RNAP.

Initiation of transcription-coupled repair characterized at single-molecule resolution

Transcription-coupled DNA repair uses components of the transcription machinery to identify DNA lesions and initiate their repair. These repair pathways are complex, so their mechanistic features remain poorly understood. Bacterial transcription-coupled repair is initiated when RNA polymerase stalled at a DNA lesion is removed by Mfd, an ATP-dependent DNA translocase. Here we use single-molecule DNA nanomanipulation to observe the dynamic interactions of Escherichia coli Mfd with RNA polymerase elongation complexes stalled by a cyclopyrimidine dimer or by nucleotide starvation. We show that Mfd acts by catalysing two irreversible, ATP-dependent transitions with different structural, kinetic and mechanistic features. Mfd remains bound to the DNA in a long-lived complex that could act as a marker for sites of DNA damage, directing assembly of subsequent DNA repair factors. These results provide a framework for considering the kinetics of transcription-coupled repair in vivo, and open the way to reconstruction of complete DNA repair pathways at single-molecule resolution.