DNA binding proteins rapidly locate their specific DNA targets through a combination of 3D and 1D diffusion mechanisms, with the 1D search involving bidirectional sliding along DNA. However, even in nucleosome-free regions, chromosomes are highly decorated with associated proteins that may block sliding. Here we investigate the ability of the abundant chromatin-associated HMGB protein Nhp6A from Saccharomyces cerevisiae to travel along DNA in the presence of other architectural DNA binding proteins using single-molecule fluorescence microscopy. We observed that 1D diffusion by Nhp6A molecules is retarded by increasing densities of the bacterial proteins Fis and HU and by Nhp6A, indicating these structurally diverse proteins impede Nhp6A mobility on DNA. However, the average travel distances were larger than the average distances between neighboring proteins, implying Nhp6A is able to bypass each of these obstacles. Together with molecular dynamics simulations, our analyses suggest two binding modes: mobile molecules that can bypass barriers as they seek out DNA targets, and near stationary molecules that are associated with neighboring proteins or preferred DNA structures. The ability of mobile Nhp6A molecules to bypass different obstacles on DNA suggests they do not block 1D searches by other DNA binding proteins.
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