Change of conformation and internal dynamics of supercoiled DNA upon binding of Escherichia coli single-strand binding protein

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Abstract
The influence of E. coli single-stranded binding (SSB) protein on the conformation and internal dynamics of pBR322 and pUC8 supercoiled DNA species was investigated by using dynamic light scattering at 632.8 and 351.1 nm, and time-resolved fluorescence polarization anisotropy of intercalated ethidium. SSB protein binds to both DNA species up to a stoichiometry that is sufficient to almost completely relax the superhelical turns. Upon saturation binding, the translational diffusion coefficients (D0) of both DNA species decrease by .apprx. 20%. Apparent diffusion coefficients (Dapp) obtained from dynamic light scattering display the well-known increase with K2 (K = scattering vector), leveling off toward a plateau value (Dplat) at high K2. For both DNA species, the difference Dplat-D0 increases upon relaxation of supercoils by SSB protein, which indicates a corresponding enhancement of the subunit mobilities in internal motions. Fluorescence polarization anisotropy measurements on free and complexed pBR322 DNA indicate a (predominantly) uniform torsional rigidity for the saturated DNA/SSB protein complex that is significantly reduced compared to the free DNA. These observations are all consistent with the notion that binding of SSB protein is accompanied by a gradual loss of supercoils and saturates when the superhelical twist is largely removed.