Introduction to DNA

This chapter provides a quick introduction to the structural properties of nucleic acids (DNA and RNA). It describes the famed double-helical structure of DNA, the more complex 3D structures adopted by RNA, and the random (possibly) twisted coil that nucleic acid can display at large scales.

Topoisomerases

This chapter discusses single-molecule approaches in the study of topoisomerases. After introducing the problem posed by DNA entanglement, it describes type I and type II topoisomerases, which solve that issue. Single-molecule assays have nailed down the different mechanisms of bacterial and eukaryotic type I topoisomerases. The properties of type II topoisomerases are then described. Single-molecule experiments have shown that they relax DNA torsion by two units, passing one dsDNA segment through a break in another segment. However, while topoII relaxes positive and negative supercoils similarly, topoIV relaxes positive supercoils quickly and processively, but negative ones slowly and distributively. This chiral discrimination is compared with the activity of the enzyme on two catenated DNA molecules. After describing single-molecule assays of the activity of gyrases, in-vivo investigations of single fluorescently labelled topoIV units in single E.coli are discussed, with concluding remarks on the future of single-molecule DNA/protein studies.

DNA Helicases

This chapter discusses the application of single-molecule approaches in the study of helicases. It describes the main helicase families and possible mechanisms of their action, and the problems of bulk experiments on helicases resulting from rehybridization of unwound strands in the wake of the enzyme; this problem is absent for RecBCD, a helicase which also possesses exonuclease activity. Two mechanisms for helicase are then discussed: passive, whereby the helicase advances by trapping DNA fork fluctuations, and active, where the DNA forces itself through the double-helical molecule. It presents two prototypical examples: RecQ, an active enzyme, and gp41, a passive one . It then shows how single-molecule experiments can be used to estimate the enzymatic step size by analysing the unwinding noise. They further led to the discovery of strand-switching. Finally, FRET experiments can be used to study the mechanisms of helicases, as demonstrated by a study of the Rep helicase.

The Mechanical Properties of Nucleic Acids

This chapter reviews models which describe the elastic properties of stretched polymers—the Kratky–Porod, Freely Jointed Chain (FJC), and Worm-Like Chain (WLC) models—and the effect of self-avoidance on results derived from these. The models are compared with double-stranded DNA (dsDNA) stretching experiments. Dynamics of a single polymer in the presence (Zimm model) or absence (Rouse model) of hydrodynamic interactions between its segments is described, and results on the dynamics of dsDNA and ssDNA of various lengths are discussed. Theoretical and experimental behaviour of twisted DNA is described, deducing the molecule’s torsional modulus and its coupling between stretching and twisting. After discussing the braiding of two DNA molecules and simulation of the twisting and stretching of DNA molecules, this chapter describes the results of stretching experiments on ssDNA and RNA, where self-avoiding and base-pairing interactions contribute to elastic behaviour.

Structural Transitions in DNA

In this chapter we discuss the various structural transitions observed on dsDNA upon twisting and stretching: the transition to denatured DNA at negative twist and to P-DNA at positive twist; the transition to S-DNA at large force and its relation with DNA melting. We discuss mechanical unzipping of DNA and show how DNA rehybridization under tension in the presence of complementary oligonucleotides can be used to sequence the molecule.

Fluorescence Spectroscopy and Microscopy Techniques

This chapter reviews the use of fluorescent methods in the study of single molecules, how the foundations of fluorescence are rooted in Einstein’s description of absorption and emission (spontaneous and stimulated), and their quantum-mechanical explanation in terms of transitions between quantized energy levels, as represented in Jablonski diagrams. It describes the non-radiative channels which compete with fluorescent emission, decrease its efficiency, and ultimately destroy the fluorescent molecule. Fluorescence Polarization Spectroscopy, FRET, and FCS are briefly presented. Without reviewing the various available fluorophores, it describes the various illumination methods used to study them, sketching super-resolution methods (STED, STORM, PALM) that have recently allowed fluorophores to be resolved to a few tens of nanometres. Finally, it describes the considerations (bandwidth, signal to noise, signal to background) used in choosing a single-molecule fluorescence detector, and the extraction of the diffusion constant of a fluorophore from the finite time, noisy traces of its positions.

Manipulating DNA

This chapter describes the various methods used to manipulate single DNA molecules and the considerations in the choice of one particular method. It starts with a description of DNA end-labelling, necessary to anchor the molecule to surfaces or beads that can be manipulated. A particular application of DNA anchoring is molecular combing, whereby the molecule is stretched on a surface by a receding meniscus. DNA rearrangements and replication bubbles can then be observed by fluorescence on these straightened molecules. It then looks at the forces at the molecular scale, which range from the smallest one due to thermal agitation, to the largest associated with breaking a covalent bond, via entropic and non-covalent bonding forces. It describes the tools used to manipulate single molecules (micro-needles, AFM cantilevers, optical, magnetic, and acoustic tweezers and traps, etc.), comparing their performances in terms of bandwidth and signal to noise (i.e., force and extension resolutions).

Conclusions

Single molecule methods have revolutionized the field of Biophysics. Much of the current applications of these methods are devoted to in vitro studies. It is our hope that the knowledge gained from these studies and described in this book will open a new vista on the in vivo investigation of cellular and physiological processes.

DNA and RNA Polymerases

This chapter discusses the application of single-molecule approaches in the study of DNA and RNA polymerases. After an introduction to DNA replication and the structure of DNA polymerases, it reviews experiments on DNA polymerization on stretched ssDNA, moving on to DNA polymerization at a stretched DNA fork (mimicking the replication fork). Next it looks at single-molecule sequencing approaches based on DNA polymerization with sequential incorporation of fluorescently labelled nucleotides, comparing with nanopore sequencing. It outlines the use of fluorescent approaches in the study of replication dynamics in vivo in single cells, then discussing transcription by RNA polymerases, the stages of transcription (open-complex, abortive initiation, transcription elongation, termination), and the general structure of RNA polymerases. It describes single-molecule experiments (using manipulation/fluorescent approaches) of the transcription stages and ends with a discussion of experiments studying the dynamics of transcription in vivo at a single locus in a eukaryotic cell with fluorescent labelling.

Single-Molecule Studies of DNA-Associated Motors

This chapter provides a quick introduction to the various uses of single-molecule approaches in the study of DNA–protein interactions. This field is a rapidly evolving one and we have not attempted to cover it in any systematic way. In the following chapters we provide examples of the type of questions addressed in the study of the interactions of DNA with three families of molecular motors: polymerases, helicases, and topoisomerases.