Book of Abstracts: Albany 2003
June 17-21 2003
Slow Kinetics of DNA/single Stranded Binding Protein SSB gp32 Interaction Studied by the Single Molecule DNA Stretching
In this work we offer a fundamentally new approach for the study of slow kinetics of DNA interaction with the ligands that affect its helix/coil equilibrium. The basic idea relies on the phenomenon of the force-induced melting of the single DNA molecule when pulled on the opposite ends (1). The melting force, fm, is an analog of the DNA melting temperature, that is sensitive to the presence of proteins. The destabilizing effect of several proteins on DNA duplex was successfully characterized before by monitoring changes in fm (2). However the equilibrium melting force is only observed when the protein/ssDNA association is fast. In the opposite case the duplex unwinding force becomes a function of the pulling rate, v. Analysis of this dependence yields kinetic parameters of protein/ss DNA interactions.
Here we develop this technique by applying it to study very strong and highly cooperative interaction of SSB bacteriophage T7 gp32 protein with DNA. This protein was studied for about 30 year, and became a paradigm for the highly cooperative duplex destabilizing protein. However some most important questions remain unanswered. For example it remains unclear why gp32 does not melt the duplex, while its mutant gp32 I* that differs in binding constant to ss DNA only 2-3 fold and retains the same high binding cooperativity w=1000 decreases the melting temperature by ~70° C. Clearly this is a kinetic effect, that was not successfully addressed by the conventional stop-flow kinetic studies. Analysis of the DNA duplex unwinding force as a function of pulling rate yields the length of ss DNA liberated in the single protein association step, Δx, that allows to estimate that ~7 bases are binding single protein. It also yields the association rate constant of the protein next to already bound one, kass. Analysis of kass as a function of protein concentration for the wild type gp32 and its two mutants offers the insight into the microscopic nature of the kinetics of this association, that consists of the two steps: the fast noncooperative association followed by the slower establishing of the protein/protein interactions. The later constant 105 s-1, was never measured before, since in the bulk studies it was always obscured by the slower protein diffusion. Our findings resolve the paradox of gp32 protein not melting DNA duplex. It also allows to explain relatively fast gp32 polymerization of ss DNA that can keep up with the fast motion of the replication fork.
We also study the very slow (minutes) relaxation of the helicity and the size distribution of the melted and helical domains in a single DNA molecule in the presence of gp32 protein and its mutants by monitoring the force relaxation to equilibrium. Analysis of the relaxation times based on our theory of the coupled relaxation of helicity and number of helix/coil boundaries offers the insight into the very slow breathing of the duplex without applied force, when very long fragments ~104 base pairs of DNA cooperatively open and close on a very long time scale.
University of Minnesota