The gene 2.5 protein (gp2.5) encoded by bacteriophage T7 binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication and recombination in the phage-infected cell. gp2.5 lowers the phage DNA melting force as measured by single molecule force spectroscopy. T7 gp2.5-⌬26C, lacking 26 acidic C-terminal residues, also reduces the melting force but at considerably lower concentrations. The equilibrium binding constants of these proteins to singlestranded DNA (ssDNA) as a function of salt concentration have been determined, and we found for example that gp2.5 binds with an affinity of (3.5 ؎ 0.6) ؋ 10 5 M ؊1 in a 50 mM Na ؉ solution, whereas the truncated protein binds to ssDNA with a much higher affinity of (7.8 ؎ 0.9) ؋ 10 7 M ؊1 under the same solution conditions. T7 gp2.5-⌬26C binding to single-stranded DNA also exhibits a stronger salt dependence than the full-length protein. The data are consistent with a model in which a dimeric gp2.5 must dissociate prior to binding to ssDNA, a dissociation that consists of a weak non-electrostatic and a strong electrostatic component.Optical tweezers have been used extensively for studying the biomechanical properties of single DNA molecules by stretching the molecules and measuring the required force for a given extension under various conditions (1-6). The mechanical work performed by stretching has an energy scale of the noncovalent interactions that hold the two DNA strands together (7) and can therefore be used to induce conversion of doublestranded DNA (dsDNA) 3 into single-stranded DNA (ssDNA).When this mechanical process is reversible, the calculated work is equal to the equilibrium melting free energy (7-9). The method of ssDNA stretching in which two strands are melted by force is referred to as force-induced melting (FIM). This method provides valuable information regarding the interaction between nucleic acids and proteins or small molecules that bind to DNA (9). In the experiments described here, a single -DNA molecule of 48,500 base pairs was stretched to extensions that were almost twice its B-form contour length, resulting in a FIM transition. Extended regions of dsDNA melt cooperatively, and the midpoint of the melting transition, F m , is analogous to the DNA melting temperature, T m , obtained in thermal melting studies. F m , like T m , is similarly affected by solution conditions such as pH, temperature, and ionic strength (5, 9, 10). DNA-binding proteins and small molecules that affect the thermal melting equilibrium of dsDNA affect the FIM transition in a similar manner (9,(11)(12)(13)(14)(15)(16)(17). One advantage of the FIM method is that DNA melting studies can be performed over a wide range of temperatures, including physiological temperature, thus avoiding protein denaturation. In addition, because the DNA is stretched during the single molecule experiment, measurements can be obtained under solution conditions that would allow the protein-DNA complex to aggregate in a bulk solution experiment.In previous ...