Binding Study of T7 Gene 2.5 Protein to Single- and Double--Stranded DNA from Single Molecule Stretching

Bacteriophage T7 gene 2.5 protein binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication, recombination, and repair. We present the first quantitative study of the thermodynamics and kinetics of equilibrium and non-equilibrium DNA helix destabilization in the presence of gp2.5 and a deletion mutant lacking 26 C-terminal amino acids that binds with higher affinity to ssDNA (gp2.5-delta26C). Our measured force-extension curves of lambda-DNA in the presence of these proteins suggest strong cooperative binding. By measuring the DNA melting force as a function of time and pulling rate, we obtained binding site size and the association constants of these proteins to ssDNA and dsDNA, over a range of salt and protein concentrations. The results are used to characterize the electrostatic interactions that determine the DNA-protein binding in each case.