Concentration-dependent exchange accelerates turnover of proteins bound to double-stranded DNA

Abstract

The multistep kinetics through which DNA-binding proteins bind their targets are heavily studied, but relatively little attention has been paid to proteins leaving the double helix. Using single-DNA stretching and fluorescence detection, we find that sequence-neutral DNA-binding proteins Fis, HU and NHP6A readily exchange with themselves and with each other. In experiments focused on the Escherichia coli nucleoid-associated protein Fis, only a small fraction of protein bound to DNA spontaneously dissociates into protein-free solution. However, if Fis is present in solution, we find that a concentration-dependent exchange reaction occurs which turns over the bound protein, with a rate of k(exch) = 6 × 10(4) M(-1)s(-1). The bacterial DNA-binding protein HU and the yeast HMGB protein NHP6A display the same phenomenon of protein in solution accelerating dissociation of previously bound labeled proteins as exchange occurs. Thus, solvated proteins can play a key role in facilitating removal and renewal of proteins bound to the double helix, an effect that likely plays a major role in promoting the turnover of proteins bound to DNA in vivo and, therefore, in controlling the dynamics of gene regulation.

Figure 1.

Schematic of instrument and sample cell. (a) DNA is tethered between the wall of a square glass capillary tube and a paramagnetic bead. Force is applied using a permanent magnet to extend the DNA in the focal plane of a fluorescence microscope (force is to the left in schematic). Images are acquired using an EMCCD camera (see ‘Materials and Methods’ section for details). (b) Schematic of sample cell assembled from glass capillaries. Force is out of the page normal to the facing surface of the cell.

Figure 2.

Sequences of Fis exchange images. DNA is tethered to a glass surface on the right with the magnetic bead on the left and an applied force of 0.7 pN. (a) Image of bead tethered by a single λ-DNA molecule prior to addition of Fis. (b) Image of complex formed on DNA by addition of 200 nM gfpFis, obtained after washing with 1 ml of buffer. (c) Same tether as in (b) after introducing 200 nM wtFis. Exchange occurs on the time-scale required to flow the new solution into the cell (∼30 s). Vertical line highlights the length difference (a two-pixel shift to the right) indicating Fis is bound. (d) Reintroduction of gfpFis replaces wtFis. Image was obtained after buffer wash as above. (e–g) Separate experiment showing exchange between gfpFis and wtHU. (h–j) Similar experiment showing exchange between gfpFis and wtNHP6A. Note the incomplete exchange evident in panels (f and i), both along the tether and the DNA coils bound to the bead, which is likely due in part to differing affinities of the solution phase and bound proteins, as discussed in the text. Scale bar (top) is ∼3 μm.

Figure 3.

Fis exchange curves and exchange rate. (a) DNA was incubated with 200 nM gfpFis, and a 1 ml buffer wash was performed prior to addition of wtFis. Images were acquired every 30 s, except for the series denoted by bowties. Note the distinctive trend to higher rates as the wtFis concentration is increased from 5 to 50 nM. Data are normalized to show the fraction of protein exchanged. Overlap of the control curves (top two curves, 30 s and 2 min acquisition intervals) indicates that bleaching contributes negligibly to the measured exchange rates. Solid lines are fits to the data. The 0 nM, 2 min fit is omitted for clarity. (b) Linear exchange rate trend as a function of wtFis concentration obtained from the curve fits in (a). Data error is standard error and error for the fit is ±1 SD.

Figure 4.

Exchange curves for different concentrations of wtHU. (a) Experiments were performed exactly as those for Figure 3 using wtHU instead of wtFis. While less pronounced than the gfpFis/wtFis exchange, there is a clear trend toward higher rates with increasing wtHU concentration. (b) The gfpFis/wtHU exchange rate trend also exhibits a linear relationship with an exchange rate constant of (2.7 ± 0.5) × 10³ M⁻¹s⁻¹. Data error is standard error and error for the fit is ±1 SD.

Figure 5.

NHP6A exchange data. (a) Complex formed from 200 nM NHP6Agfp. (b) Addition of 200 nM wtNHP6A resulted in complete exchange with the bound NHP6Agfp. (c) Re-addition of 200 nM NHP6Agfp resulted in exchange with bound wtNHP6A. (d) NHP6A exchange curves. Each curve is the average of several data sets. Because most of the exchange occurs so quickly (i.e. before the first image could be acquired), the data is presented in terms of absolute fluorescence showing a clear acceleration of off rate after addition of 400 nM wtNHP6A (bottom curve) compared to 0 nM wtNHP6A (top curve). The error in the initial fluorescence point is the standard error of the mean for several data sets.

Figure 6.

Non-specific DNA binding by gfpFis and wtFis evaluated by gel mobility shift experiments. gfpFis (a) and wtFis (b) were incubated with a 150 bp ³²P-labeled fragment from the S. cerevisiae MET14 gene and subjected to electrophoresis in a native 5% polyacrylamide gel. 0 designates no added protein followed by 2-fold increasing amounts of protein beginning at 1.3 nM for gfpFis and 1.1 nM for wtFis. Complexes containing from 1 to 6–8 dimers of Fis are formed with increasing amounts of added protein in both cases. The calculated K_d for the first bound complex was 3.1 ± 1.4 and 1.7 ± 0.5 nM and for the fully-coated DNA complex [seven Fis dimers, see ref. (14)] was 27.5 ± 7.8 and 32.0 ± 3.6 nM for gfpFis (n = 3) and wtFis (n = 5), respectively. At ≥70 nM both proteins also form a high-order complex referred to as the low-mobility complex (LMC). The slower relative migrations of the gfpFis complexes are consistent with the MW difference of the dimeric proteins: 76.6 kDa for gfpFis and 22.8 kDa for wtFis.