When single DNA molecules are stretched, the measurement of the resulting force as a function of extension has yielded new information about the physical, chemical, and biological properties of these important molecules. It has been shown that double-stranded DNA molecules undergo a force-induced melting transition at high forces. Force-extension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review our studies of DNA force-induced melting in the presence of the classical single-stranded DNA binding protein, gene 32 protein. Bacteriophage T4 gene 32 protein (gp32) is a well studied representative of a large class of single-stranded DNA binding proteins, which are essential for the replication, recombination and repair of DNA. We have developed several new single molecule methods, which when applied to gp32, have led to significant new insights about this protein's structure-function relationships. We discuss a technique for measuring Kss, the association constant of these proteins to ssDNA, which we can determine over a large range of salt concentrations not available to bulk binding studies. In addition, we have measured the noncooperative association constants (Kds,) of the weak but biologically-significant interaction with double-stranded DNA as a function of salt concentration for full-length protein and *I, a truncation of gp32 lacking the 48-residue C-terminal domain. Our results have led to a quantitative model for the salt dependence of protein binding, which we postulate to be regulated by a salt-dependent conformational change within the protein involving the C-terminal domain. With this new force spectroscopy technique, we have obtained binding rates and binding free energies for these interactions under a broad range of conditions. Our methodologies should have useful applications in many areas of DNA-protein interactions.
- Association constant
- DNA melting
- N-terminal domain (NTD)
- Non-equilibrium force spectroscopy
- gp32 binding kinetics