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.

The elastic properties of single stranded (ss)DNA, studied by pulling on an isolated molecule, are shown to agree with a recent model of ssDNA that takes into account base pairings and screened electrostatic repulsion of the phosphodiester backbone. By an appropriate physicochemical treatment, the pairing interactions were suppressed and ssDNA used as an experimental model for a generic polyelectrolyte. The elastic behavior of such an altered ssDNA deviates strongly from the behavior of an ideal polymer. This deviation is shown to result from the elasticity of the chain and its electrostatic selfavoiding interactions.

DNA helicases are enzymes capable of unwinding double-stranded DNA (dsDNA) to provide the single-stranded DNA template required in many biological processes. Among these, UvrD, an essential DNA repair enzyme, has been shown to unwind dsDNA while moving 3-5 on one strand. Here, we use a single-molecule manipulation technique to monitor real-time changes in extension of a single, stretched, nicked dsDNA substrate as it is unwound by a single enzyme. This technique offers a means for measuring the rate, lifetime, and processivity of the enzymatic complex as a function of ATP, and for estimating the helicase step size. Strikingly, we observe a feature not seen in bulk assays: unwinding is preferentially followed by a slow, enzyme-translocation-limited rezipping of the separated strands rather than by dissociation of the enzymatic complex followed by quick rehybridization of the DNA strands. We address the mechanism underlying this phenomenon and propose a fully characterized model in which UvrD switches strands and translocates backwards on the other strand, allowing the DNA to reanneal in its wake.helicase ͉ DNA replication ͉ DNA repair ͉ magnetic tweezers A lthough helicases are essential molecular motors, their precise mechanism is only partially known. These motors translocate along DNA while stripping off one strand of the double helix (1, 2). Whereas a large number of helicases involved in DNA repair and recombination are either monomeric or dimeric, replicative helicases typically form processive, hexameric entities surrounding one or both strands. The process of separating the two strands of DNA is often described as the translocation of the enzyme on one strand, which defines the directionality of the process, whereas the displacement of the other strand is accomplished actively, if the helicase melts the base pairs, or, passively, if the helicase moves forward as the bases transiently unpair. Observing the activity of a single helicase unwinding a double helix yields valuable information on the enzymatic dynamics, as has been the case for other molecular motors such as kinesin and myosin (3).UvrD (720 aa, molecular mass ϭ 82 kDa), a member of the helicase SF1 superfamily (which includes PcrA and Rep), plays a crucial role in nucleotide excision repair and methyl-directed mismatch repair (4-8) and is required for the replication of several plasmids (9). It has been shown to initiate unwinding from a 3Ј end single-stranded DNA (ssDNA) tail, a gap, or a nick and to translocate along ssDNA in a 3Ј-5Ј direction (10-12). The purpose of this study is to investigate the mechanochemistry of UvrD-catalyzed DNA unwinding at the single-molecule level, yielding more direct insight into its enzymatic activity by avoiding the inherent averaging of bulk assays. We present real-time measurements of the unwinding rate, lifetime, and number of base pairs unwound, as well as an estimate of the step size. In addition, we observe that an unwinding event can be followed by an enzyme-translocation-limited rehybridization of the o...