Single molecule analysis of Thermus thermophilus SSB protein dynamics on single-stranded DNA

Abstract

Single-stranded (ss) DNA binding (SSB) proteins play central roles in DNA replication, recombination and repair in all organisms. We previously showed that Escherichia coli (Eco) SSB, a homotetrameric bacterial SSB, undergoes not only rapid ssDNA-binding mode transitions but also one-dimensional diffusion (or migration) while remaining bound to ssDNA. Whereas the majority of bacterial SSB family members function as homotetramers, dimeric SSB proteins were recently discovered in a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Here we show, using single-molecule fluorescence resonance energy transfer (FRET), that homodimeric bacterial SSB from Thermus thermophilus (Tth) is able to diffuse spontaneously along ssDNA over a wide range of salt concentrations (20-500 mM NaCl), and that TthSSB diffusion can help transiently melt the DNA hairpin structures. Furthermore, we show that two TthSSB molecules undergo transitions among different DNA-binding modes while remaining bound to ssDNA. Our results extend our previous observations on homotetrameric SSBs to homodimeric SSBs, indicating that the dynamic features may be shared among different types of SSB proteins. These dynamic features of SSBs may facilitate SSB redistribution and removal on/from ssDNA, and help recruit other SSB-interacting proteins onto ssDNA for subsequent DNA processing in DNA replication, recombination and repair.

Figure 1.

smFRET assays that report the conformations of TthSSB-bound ssDNA. (A) A schematic illustration of our sm FRET experimental design for TthSSB. A partial duplex DNA substrate [(dT)₆₀] was immobilized on a PEG-coated surface. Cy5 and Cy3 were attached to the ss–dsDNA junction and the end of the ssDNA overhang, respectively. (B) The smFRET histograms for (dT)₆₀ DNA alone and TthSSB binding to (dT)₆₀ at different salt concentrations. TthSSB proteins were loaded onto (dT)₆₀ under high salt condition (500 mM NaCl) and unbound proteins were removed before data acquisition (see also Materials and methods section).

Figure 2.

Protein diffusion detection assays based on DNA hybridization. (A–C) Representative single-molecule time traces (A), smFRET histograms (B) and average cross-correlation curves (C), for (dT)_60+12m DNA alone and TthSSB-bound (dT)_60+12m with and without the hybridization to the 12-nt mixture sequence ssDNA region. FRET fluctuations beyond measurement noise were detected only when the 12-nt extension is available for TthSSB binding. Unbound proteins were removed before data acquisition such that the FRET flucatuations reflect only the repositioning of the bound TthSSB along (dT)_60+12m. For simplification, the 18-bp duplex DNA region is not shown for TthSSB-bound (dT)_60+12m in (A). The solid line in (C) is a fit to a single exponential function.

Figure 3.

TthSSB appears to be positioned near the center of the (dT)_60+n ssDNA overhangs though rapid diffusion. (A) A schematic illustration of our experimental design for (dT)_60+n (n = 0, 4, 8, 12 or 16). Cy3 and Cy5 were attached to the ss–dsDNA junction and the middle of the ssDNA overhang, respectively, separated by (dT)₆₀. (B) The smFRET histograms of (dT)_60+n DNA in the absence of proteins, obtained at 500 mM NaCl. (C) smFRET histograms for TthSSB-bound (dT)_60+n, obtained at 500 mM NaCl, showing a single narrow FRET peak. (D) Average cross-correlation curves for TthSSB-bound (dT)_60+n, obtained at 500 mM NaCl, indicating no significant FRET fluctuations. (E) The FRET value at the FRET peak position versus the n value in (dT)_60+n, obtained at 20, 100 or 500 mM NaCl. (F) The FRET value at the FRET peak position versus the salt concentration for different (dT)_60+n substrates. TthSSB proteins were loaded onto (dT)₆₀ under high salt condition (500 mM NaCl) and unbound proteins were removed before the buffer containing a lower NaCl concentration was added to the sample chamber.

Figure 4.

Lower temperatures slow TthSSB diffusion on (dT)₆₀₊₄. (A) Representative single-molecule time traces for TthSSB-bound (dT)₆₀₊₄ obtained at different temperatures (23, 18, 14 and 11℃C) and at 100 mM NaCl. (B) A schematic illustration of our experimental design, showing how TthSSB diffusion may result in FRET fluctuations. (C) Average cross-correlation curves for TthSSB-bound (dT)₆₀₊₄ obtained at different temperatures and at 100 mM NaCl. More significant FRET fluctuations beyond measurement noise were detected at lower temperatures. The time scales of the FRET fluctuations were determined from fits to a single exponential function (solid lines). Unbound proteins were removed before data acquisition. (D) Arrhenius plot of apparent rates as a function of 1/T.

Figure 5.

TthSSB diffusion can transiently destabilize short DNA hairpin structures. (A and B) Representative single-molecule time traces (A) and average cross-correlation curves (B), for (dT)_65+hp+3 alone and TthSSB-bound (dT)_65+hp+3, obtained at 500 mM NaCl. Unbound proteins were removed before data acquisition. The solid line in (B) is a fit to a single exponential function.

Figure 6.

Transitions between different binding modes for TthSSB binding to (dT)₆₀ in the presence of proteins in solution. (A) Representative sm FRET-time traces for TthSSB-bound (dT)₆₀, obtained at 500 and 100 mM NaCl when 10 nM TthSSB were present, and those obtained at 20 mM NaCl when 10 or 1 nM TthSSB were present in the sample camber. 1 nM TthSSB were first incubated with the surface-tethered (dT)₆₀ in 500 mM NaCl, followed by a buffer wash to remove unbound TthSSB proteins. The buffer containing the indicated TthSSB and NaCl concentration was then injected into the chamber for data acquisition. ΔT_H and ΔT_L represent the durations for the high and low FRET state, respectively. (B) Transtion rates of the interconversions between high and low FRET state (1/ΔT_H and 1/ΔT_L represent the high to low FRET-state transiton and the low to high FRET-state transition, respectively), as a function of the TthSSB concentration. Solid lines are the linear fits for high to low FRET-state transition rate as a function of TthSSB concentration.

Figure 7.

Transitions between different binding modes for TthSSB binding to (dT)₆₀₊₁₆ in the presence of proteins in solution. (A) Representative sm FRET-time traces for TthSSB-bound (dT)₆₀₊₁₆, obtained at 100 mM NaCl and when 0, 0.5, 1, 2 or 4 nM TthSSB were present in the sample camber. ΔT_dynamic and ΔT_static represent the durations for the ‘dynamic’ and ‘static’ states, respectively. (B) Association and dissociation rates of the second TthSSB to/from (dT)₆₀₊₁₆ (1/ΔT_static and 1/ΔT_dynamic, respectively), as a function of the TthSSB concentration. (C) Hidden Markov model (HMM)-derived idealized FRET trajectory (red) superimposed on the FRET-time trace (blue), for a selected time period during which the molecule is in the ‘dynamic’ state. Two FRET states were determined from the HMM fit (∼0.3 and ∼0.6 FRET). (D) FRET-efficiency distributions within the ‘dynamic’ state at different TthSSB concentrations (averaged from >50 molecules). Solid lines are the fits to a double-Gaussian function. (E) Transition rates between the 0.3 and 0.6 FRET state, determined from the HMM fit at different TthSSB concentrations.

Figure 8.

Model of TthSSB dynamics on ssDNA. (dT)₆₀₊₁₆ is illustrated as the ssDNA template for TthSSB binding. For simplification, only the ssDNA region is shown in the cartoon for TthSSB-bound (dT)₆₀₊₁₆.