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Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways.

Suksombat S, Khafizov R, Kozlov AG, Lohman TM, Chemla YR - Elife (2015)

Bottom Line: Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated.The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled.Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Center for the Physics of Living Cells, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States.

ABSTRACT
Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA.

No MeSH data available.


Related in: MedlinePlus

Occurrence probability of SSB wrapping intermediates.Extension change distributions (left panels) of many SSB wrapping events obtained from force-ramp experiments (1 pN) and constant force experiments (2–10 pN). Individual wrapping intermediates are analyzed and assigned to corresponding SSB binding modes based on Figure 3C. At all tensions, the probability of each SSB binding modes (right panels, color bars) is derived from the area under the distributions. The model (black circles, ‘Materials and methods’) obtained from the energy landscape in Figure 6 matches well with the experimentally derived probabilities.DOI:http://dx.doi.org/10.7554/eLife.08193.021
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fig6s1: Occurrence probability of SSB wrapping intermediates.Extension change distributions (left panels) of many SSB wrapping events obtained from force-ramp experiments (1 pN) and constant force experiments (2–10 pN). Individual wrapping intermediates are analyzed and assigned to corresponding SSB binding modes based on Figure 3C. At all tensions, the probability of each SSB binding modes (right panels, color bars) is derived from the area under the distributions. The model (black circles, ‘Materials and methods’) obtained from the energy landscape in Figure 6 matches well with the experimentally derived probabilities.DOI:http://dx.doi.org/10.7554/eLife.08193.021

Mentions: As described in the text, each peak in the histograms of extension change vs force in Figure 2 was assigned a particular wrapping state i, as detailed in Figure 3. We determined the probability pi(F) from the ratio of the area under the peak to the total area in the histogram at force F, (Figure 6—figure supplement 1). From Equation 9, we determined the free energy difference between pairs of states, evaluating ΔGstretch(F) from the area between curves of extension vs force for the two wrapping states i and j according to Equation 2. Since some of the same states were populated at different forces, we obtained several estimates of the same free energy differences. All yielded consistent values, which were averaged together and used to calculate a standard error. Setting the free energy of the unwrapped state G0 = 0, the free energy associated with each state was calculated to be G17 = −6.80 ± 0.82 kBT, G35 = −15.38 ± 0.57 kBT, G56 = −20.39 ± 0.83 kBT, and G65 = −21.11 ± 0.83 kBT. The corresponding energy landscape is presented in Figure 6.


Structural dynamics of E. coli single-stranded DNA binding protein reveal DNA wrapping and unwrapping pathways.

Suksombat S, Khafizov R, Kozlov AG, Lohman TM, Chemla YR - Elife (2015)

Occurrence probability of SSB wrapping intermediates.Extension change distributions (left panels) of many SSB wrapping events obtained from force-ramp experiments (1 pN) and constant force experiments (2–10 pN). Individual wrapping intermediates are analyzed and assigned to corresponding SSB binding modes based on Figure 3C. At all tensions, the probability of each SSB binding modes (right panels, color bars) is derived from the area under the distributions. The model (black circles, ‘Materials and methods’) obtained from the energy landscape in Figure 6 matches well with the experimentally derived probabilities.DOI:http://dx.doi.org/10.7554/eLife.08193.021
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4582245&req=5

fig6s1: Occurrence probability of SSB wrapping intermediates.Extension change distributions (left panels) of many SSB wrapping events obtained from force-ramp experiments (1 pN) and constant force experiments (2–10 pN). Individual wrapping intermediates are analyzed and assigned to corresponding SSB binding modes based on Figure 3C. At all tensions, the probability of each SSB binding modes (right panels, color bars) is derived from the area under the distributions. The model (black circles, ‘Materials and methods’) obtained from the energy landscape in Figure 6 matches well with the experimentally derived probabilities.DOI:http://dx.doi.org/10.7554/eLife.08193.021
Mentions: As described in the text, each peak in the histograms of extension change vs force in Figure 2 was assigned a particular wrapping state i, as detailed in Figure 3. We determined the probability pi(F) from the ratio of the area under the peak to the total area in the histogram at force F, (Figure 6—figure supplement 1). From Equation 9, we determined the free energy difference between pairs of states, evaluating ΔGstretch(F) from the area between curves of extension vs force for the two wrapping states i and j according to Equation 2. Since some of the same states were populated at different forces, we obtained several estimates of the same free energy differences. All yielded consistent values, which were averaged together and used to calculate a standard error. Setting the free energy of the unwrapped state G0 = 0, the free energy associated with each state was calculated to be G17 = −6.80 ± 0.82 kBT, G35 = −15.38 ± 0.57 kBT, G56 = −20.39 ± 0.83 kBT, and G65 = −21.11 ± 0.83 kBT. The corresponding energy landscape is presented in Figure 6.

Bottom Line: Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated.The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled.Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA.

View Article: PubMed Central - PubMed

Affiliation: Department of Physics, Center for the Physics of Living Cells, Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, United States.

ABSTRACT
Escherichia coli single-stranded (ss)DNA binding (SSB) protein mediates genome maintenance processes by regulating access to ssDNA. This homotetrameric protein wraps ssDNA in multiple distinct binding modes that may be used selectively in different DNA processes, and whose detailed wrapping topologies remain speculative. Here, we used single-molecule force and fluorescence spectroscopy to investigate E. coli SSB binding to ssDNA. Stretching a single ssDNA-SSB complex reveals discrete states that correlate with known binding modes, the likely ssDNA conformations and diffusion dynamics in each, and the kinetic pathways by which the protein wraps ssDNA and is dissociated. The data allow us to construct an energy landscape for the ssDNA-SSB complex, revealing that unwrapping energy costs increase the more ssDNA is unraveled. Our findings provide insights into the mechanism by which proteins gain access to ssDNA bound by SSB, as demonstrated by experiments in which SSB is displaced by the E. coli recombinase RecA.

No MeSH data available.


Related in: MedlinePlus