Architectural DNA-binding proteins function to regulate diverse DNA reactions and have the defining property of significantly changing DNA conformation. Although the 1D movement along DNA by other types of DNA-binding proteins has been visualized, the mobility of architectural DNA-binding proteins on DNA remains unknown. Here, we applied single-molecule fluorescence imaging on arrays of extended DNA molecules to probe the binding dynamics of three structurally distinct architectural DNA-binding proteins: Nhp6A, HU, and Fis. Each of these proteins was observed to move along DNA, and the salt concentration independence of the 1D diffusion implies sliding with continuous contact to DNA. Nhp6A and HU exhibit a single sliding mode, whereas Fis exhibits two sliding modes. Based on comparison of the diffusion coefficients and sizes of many DNA binding proteins, the architectural proteins are categorized into a new group distinguished by an unusually high free-energy barrier for 1D diffusion. The higher free-energy barrier for 1D diffusion by architectural proteins can be attributed to the large DNA conformational changes that accompany binding and impede rotation-coupled movement along the DNA grooves.

Architectural DNA-binding proteins (ADBPs) are abundant constituents of eukaryotic or bacterial chromosomes that bind DNA promiscuously and function in diverse DNA reactions. They generate large conformational changes in DNA upon binding yet can slide along DNA when searching for functional binding sites. Here we investigate the mechanism by which ADBPs diffuse on DNA by single-molecule analyses of mutant proteins rationally chosen to distinguish between rotation-coupled diffusion and DNA surface sliding after transient unbinding from the groove(s). The properties of yeast Nhp6A mutant proteins, combined with molecular dynamics simulations, suggest Nhp6A switches between two binding modes: a static state, in which the HMGB domain is bound within the minor groove with the DNA highly bent, and a mobile state, where the protein is traveling along the DNA surface by means of its flexible N-terminal basic arm. The behaviors of Fis mutants, a bacterial nucleoid-associated helix-turn-helix dimer, are best explained by mobile proteins unbinding from the major groove and diffusing along the DNA surface. Nhp6A, Fis, and bacterial HU are all near exclusively associated with the chromosome, as packaged within the bacterial nucleoid, and can be modeled by three diffusion modes where HU exhibits the fastest and Fis the slowest diffusion.