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We describe the mechanical separation of the two complementary strands of a single molecule of bacteriophage DNA. The 3 and 5 extremities on one end of the molecule are pulled progressively apart, and this leads to the opening of the double helix. The typical forces along the opening are in the range of 10-15 pN. The separation force signal is shown to be related to the local GC vs. AT content along the molecule. Variations of this content on a typical scale of 100-500 bases are presently detected. Mechanical force at the molecular level is involved in the action of many enzymes. This is the case for the processes of replication or transcription in which enzymes translocate processively with respect to DNA. Such translocation occurs unidirectionally over long segments of DNA, and the enzymatic machinery has to develop a force against a number of impediments: the disruption of complementary base pairs, the possible attachments of the DNA or the enzymes to cellular components, structural proteins that coat DNA and have to be displaced, topological constraint, and viscous friction. The force necessary to stop a transcribing Escherichia coli polymerase recently has been measured (1). In the case of replication (2), the DNA double helix is opened, and two daughter strands are formed. The opening may be associated to the translocation mechanism of the polymerase itself or may be assisted by accessory proteins like helicases or single-strand binding proteins. Moreover, because the strands are intertwined, strand separation is coupled to a local rotation. Topological constraints are resolved by topoisomerases (3, 4).Long before the enzymes associated to replication were known, a simple model had been considered (5) in which the mechanism of unwinding the strands during replication is coupled to rotation of the whole molecule. A molecular configuration with a Y shape had been proposed in which the vertical part is the parent helix, and the two arms are the separated strands that get replicated. As replication proceeds, a ''speedometer cable'' rotation motion was proposed for all three branches of the Y.We have set up an experiment to measure directly the forces involved in the elementary process of mechanical strand separation, with no enzyme present. The experiment presented here is approaching the Levinthal and Crane (5) configuration.Force measurement on single molecules of DNA is an emerging field (6-13). For typical molecular interactions involving biomolecules, the forces involved are small (sub-picoNewton to 10s of picoNewtons [pN ϭ 10 Ϫ12 N]). For this reason, sensitive measuring devices (14-17) such as optical tweezers, soft microneedles, or levers of atomic force microscopes have been used.

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