Genome replication and maintenance occurs through the collective action of proteins that operate on single-stranded DNA (ssDNA). All cells express single-stranded DNA binding proteins (SSBs), which prevent errors by sequestering ssDNA with high-affinity, keeping it free from transient structures and protecting it from unwanted chemical modification. SSBs must be easily repositioned, or else risk stalling DNA replication and repair processes. How does a protein simulataneously bind DNA tightly yet diffuse rapidly?
Through a set of extensive all-atom molecular dynamics (MD) simulations, we have elucidated the molecular mechanism of SSB association with ssDNA. First, we showed that the same SSB-ssDNA complex can both spontaneously rearrange its structure and maintain its stable conformation depending on whether it is surrounded by physiological solution or a protein-crystal environment. Next, we probed the local interaction between ssDNA and SSB through simulations of mechanical unraveling of the SSB-ssDNA assemblies and simulations of spontaneous association of ssDNA fragments with SSB. We found mechanical unraveling of ssDNA to be highly stochastic while stalling at reproducible sites along the SSB surface—the same sites that bind ssDNA with high affinity in our DNA association simulations. Finally, we directly observe microscopic events that constitute the elementary steps of SSB diffusion along ssDNA: formation of ssDNA bulges and their stochastic motion between high-affinity sites of SSB. To the best of our knowledge, this is the first direct observation of a diffusive motion within a protein–DNA complex by an equilibrium all-atom MD simulation. Based on the data derived from the three complimentary approaches, we conclude that SSB–ssDNA assemblies are formed by loosely associated stretches of ssDNA pinned to high-affinity spots at the SSB surface. The rate of SSB diffusion along ssDNA is likely determined by dissociation of ssDNA from these spots.