Molecular mechanism of DNA association with single-stranded DNA binding protein

Christopher Maffeo, and Aleksei Aksimentiev
Nucleic Acids Research 45(21) 12125-12139 (2017)
DOI:10.1093/nar/gkx917  BibTex

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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.

Abstract

During DNA replication, the single-stranded DNA binding protein (SSB) wraps single-stranded DNA (ssDNA) with high affinity to protect it from degradation and prevent secondary structure formation. Although SSB binds ssDNA tightly, it can be repositioned along ssDNA to follow the advancement of the replication fork. Using all-atom molecular dynamics simulations, we characterized the molecular mechanism of ssDNA association with SSB. Placed in solution, ssDNA–SSB assemblies were observed to change their structure spontaneously; such structural changes were suppressed in the crystallographic environment. Repeat simulations of the SSB–ssDNA complex under mechanical tension revealed a multitude of possible pathways for ssDNA to come off SSB punctuated by prolonged arrests at reproducible sites at the SSB surface. Ensemble simulations of spontaneous association of short ssDNA fragments with SSB detailed a three-dimensional map of local affinity to DNA; the equilibrium amount of ssDNA bound to SSB was found to depend on the electrolyte concentration but not on the presence of the acidic tips of the SSB tails. Spontaneous formation of ssDNA bulges and their diffusive motion along SSB surface was directly observed in multiple 10-µs-long simulations. Such reptation-like motion was confined by DNA binding to high-affinity spots, suggesting a two-step mechanism for SSB diffusion.

DNA bound to single-stranded DNA binding protein (SSB) is seen to adopt many microscopic configurations when immersed in a solution. The animation depicts a 500 ns equilibration simulation of the SSB65 complex in a 160 mM electrolyte solution. The DNA is depicted using green van der Waals (vdW) spheres, and the protein is depicted as a white molecular surface. Water and ions are not shown. The complex is rotated about the vertical axis during the animation.

The configuration of DNA bound to SSB in the crystallographic environment is stabilized by contacts with adjacent proteins. The animation illustrates a nearly 500 ns equilibration simulation of an SSB–ssDNA complex in the crystallographic environment. The unit cell for the crystal environment contains four SSB oligomers (light blue) and eight 35 nt ssDNA fragments (green) submerged in a 100 mM electrolyte solution. At the onset of the simulation, SSB and ssDNA atoms that were resolved in the crystal structure had the crystallographic coordinates (PDB accession code: 1EYG). One crystallographic unit cell is highlighted using green vdW spheres for DNA and molecular surfaces for the SSB proteins. Several copies of the crystallographic unit cells are depicted in the background using a less detailed molecular surface representation. Water and ions are not shown. 

Force-induced rupture of an SSB ssDNA complex stalls at arginine-rich sites. The animation depicts the rupture of a SSB65 complex. The free ends of the springs were moved away from the protein along the vertical axis at a constant rate. Atoms forming the DNA backbone and base are depicted as green and light-green vdW spheres, respectively. Each protein atom is depicted as a vdW sphere with a color selected from four shades of blue or cyan, depending on which monomer the atom belongs to. Three views of the system are shown simultaneously: two that track the terminal nucleotides bound to the protein on the 3′ (top) and 5′ (bottom) ends, and one view that tracks the center of the DNA bound to the protein (right). Arginine residues of the DNA binding groove between loops L11′ and L23 and the N-terminal arginine-rich patch are shown using orange and yellow vdW spheres, respectively. In the lower-right quadrant, the index of the first (5′ end) and last (3′ end) nucleotide of the ssDNA fragment bound to SSB are shown in a plot. The nucleotides are numbered in ascending order from 5′ to 3′ ends of ssDNA. The simulation was performed after approximately 100 ns of unbiased equilibration and lasted 425 ns.

The acidic C-terminal tips of SSB cannot compete with ssDNA for binding the SSB surface. The animation shown one of six simulations lasting 340 ns that characterized competitive binding of ssDNA and C-terminus peptide fragments for SSB. The systems contained fifteen dT3 fragments, eight 3-amino acid peptide fragments, and a solvent comprising only water and neutralizing concentration (40 mM) of Na+ counterions. The DNA is depicted using vdW spheres, the protein is depicted as a white molecular surface and the C-terminal acidic tip peptide fragments are shown as purple vdW spheres.

Long timescale simulations of SSB–ssDNA complexes show large scale motion of the DNA. The animation illustrates a 10 μs MD trajectory of SSB65 in an electrolyte containing 50 mM K+. The DNA is depicted using vdW spheres, and the protein is depicted as a white molecular surface. 

The electrostatic environment around the SSB complex has surprisingly little effect on the ten-microsecond timescale. This animation illustrates an MD trajectory of SSB65 in a 1100 mM KCl electrolyte. The DNA is depicted using vdW spheres, and the protein is depicted as a white molecular surface.

The acidic tips in the SSB-GG mutant spend much time adsorbed to the groove at the dimer–dimer interface of SSB. The animation depicts a 10 μs MD trajectory of the SSB-GG mutant in a 170 mM KCl solution. The DNA is depicted using vdW spheres, the protein is depicted as a white molecular surface and the C-terminal acidic tips are shown as purple vdW spheres.

Small bulges of DNA on the surface of SSB were seen to move along the SSB surface, consistent with the reptation model of diffusion. The animation highlights a reptation event during the simulation of SSB65 in an electrolyte containing 50 mM K+. VdW spheres depict SSB (light blue) and DNA backbone atoms. Each nucleotide is depicted using a different color. Water, ions, and the DNA bases are not shown for clarity. The animation shows the entire 10 μs trajectory.

Large scale motion of a stretch of ssDNA bound to the SSB surface between arginine rich regions of hte SSB surface. The animation illustrates concerted motion of an 8 nucleotides ssDNA fragment during the simulation of the SSB-GG mutant in 170 mM KCl. The nucleotides were seen to move across the SSB surface orthogonal to the wrapping path and returning to the original location. One end of these nucleotides remained bound to a DBG (orange) and the other end remained bound to an N-terminal arginine patch (yellow). VdW spheres depict the SSB atoms (blue to white) and DNA backbone atoms (green). The animation shows a 1 μs excerpt of the 10 μs trajectory starting after ~6 μs