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Mechanical force antagonizes the inhibitory effects of RecX on RecA filament formation in Mycobacterium tuberculosis.

Le S, Chen H, Zhang X, Chen J, Patil KN, Muniyappa K, Yan J - Nucleic Acids Res. (2014)

Bottom Line: However, applying larger forces antagonized the inhibitory effects of MtRecX, and a partially de-polymerized MtRecA filament could re-polymerize in the presence of MtRecX, which cannot be explained by previous models.Theoretical analysis of force-dependent conformational free energies of naked ssDNA and RecA nucleoprotein filament suggests that mechanical force stabilizes RecA filament, which provides a possible mechanism for the observation.As the antagonizing effect of force on the inhibitory function of RecX takes place in a physiological range; these findings broadly suggest a potential mechanosensitive regulation during homologous recombination.

View Article: PubMed Central - PubMed

Affiliation: Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore Department of Physics, National University of Singapore, Singapore 117542, Singapore.

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Force-dependent conformational free energy difference between RecA filament and ssDNA. The force-dependent conformational free energy difference per nucleotide between RecA filament and ssDNA, , is negative in the force range of 0–90 pN and shows a non-monotonic force dependence.  is obtained by WLC fitting and  is obtained by cubic spline interpolation to experimental data of RecA filament and naked ssDNA in Figure 1B, respectively. Insert shows the experimentally measured polymerization (circles) and de-polymerization (tri-angles) rates of MtRecA filament at multiple forces. Shadow indicates the force range at which no observable de-polymerization of the MtRecA filaments for > 1000 s (Supplementary Figure S10). Note that the polymerization rate was not measured for force <10 pN to avoid formation of secondary structure that may hamper the polymerization of MtRecA on ssDNA.
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Figure 4: Force-dependent conformational free energy difference between RecA filament and ssDNA. The force-dependent conformational free energy difference per nucleotide between RecA filament and ssDNA, , is negative in the force range of 0–90 pN and shows a non-monotonic force dependence. is obtained by WLC fitting and is obtained by cubic spline interpolation to experimental data of RecA filament and naked ssDNA in Figure 1B, respectively. Insert shows the experimentally measured polymerization (circles) and de-polymerization (tri-angles) rates of MtRecA filament at multiple forces. Shadow indicates the force range at which no observable de-polymerization of the MtRecA filaments for > 1000 s (Supplementary Figure S10). Note that the polymerization rate was not measured for force <10 pN to avoid formation of secondary structure that may hamper the polymerization of MtRecA on ssDNA.

Mentions: The force-dependent conformational free energies of RecA filament and ssDNA can be calculated from their respective force-extension curves and through relations: and . were directly measured in our buffered reaction solution conditions (Figure 1B), which were used to calculate per nucleotide ssDNA. As is negative up to 90 pN, force in this range facilitates RecA polymerization by reducing the free energy cost for polymerization and is optimized in force range of 20–25 pN (Figure 4). The equilibrium between RecA polymerization and de-polymerization depends on the rates of polymerization and de-polymerization, kon and koff, respectively, through . Therefore, the effect of force on shifting the equilibrium must be through either increasing kon or decreasing koff, or both.


Mechanical force antagonizes the inhibitory effects of RecX on RecA filament formation in Mycobacterium tuberculosis.

Le S, Chen H, Zhang X, Chen J, Patil KN, Muniyappa K, Yan J - Nucleic Acids Res. (2014)

Force-dependent conformational free energy difference between RecA filament and ssDNA. The force-dependent conformational free energy difference per nucleotide between RecA filament and ssDNA, , is negative in the force range of 0–90 pN and shows a non-monotonic force dependence.  is obtained by WLC fitting and  is obtained by cubic spline interpolation to experimental data of RecA filament and naked ssDNA in Figure 1B, respectively. Insert shows the experimentally measured polymerization (circles) and de-polymerization (tri-angles) rates of MtRecA filament at multiple forces. Shadow indicates the force range at which no observable de-polymerization of the MtRecA filaments for > 1000 s (Supplementary Figure S10). Note that the polymerization rate was not measured for force <10 pN to avoid formation of secondary structure that may hamper the polymerization of MtRecA on ssDNA.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4231760&req=5

Figure 4: Force-dependent conformational free energy difference between RecA filament and ssDNA. The force-dependent conformational free energy difference per nucleotide between RecA filament and ssDNA, , is negative in the force range of 0–90 pN and shows a non-monotonic force dependence. is obtained by WLC fitting and is obtained by cubic spline interpolation to experimental data of RecA filament and naked ssDNA in Figure 1B, respectively. Insert shows the experimentally measured polymerization (circles) and de-polymerization (tri-angles) rates of MtRecA filament at multiple forces. Shadow indicates the force range at which no observable de-polymerization of the MtRecA filaments for > 1000 s (Supplementary Figure S10). Note that the polymerization rate was not measured for force <10 pN to avoid formation of secondary structure that may hamper the polymerization of MtRecA on ssDNA.
Mentions: The force-dependent conformational free energies of RecA filament and ssDNA can be calculated from their respective force-extension curves and through relations: and . were directly measured in our buffered reaction solution conditions (Figure 1B), which were used to calculate per nucleotide ssDNA. As is negative up to 90 pN, force in this range facilitates RecA polymerization by reducing the free energy cost for polymerization and is optimized in force range of 20–25 pN (Figure 4). The equilibrium between RecA polymerization and de-polymerization depends on the rates of polymerization and de-polymerization, kon and koff, respectively, through . Therefore, the effect of force on shifting the equilibrium must be through either increasing kon or decreasing koff, or both.

Bottom Line: However, applying larger forces antagonized the inhibitory effects of MtRecX, and a partially de-polymerized MtRecA filament could re-polymerize in the presence of MtRecX, which cannot be explained by previous models.Theoretical analysis of force-dependent conformational free energies of naked ssDNA and RecA nucleoprotein filament suggests that mechanical force stabilizes RecA filament, which provides a possible mechanism for the observation.As the antagonizing effect of force on the inhibitory function of RecX takes place in a physiological range; these findings broadly suggest a potential mechanosensitive regulation during homologous recombination.

View Article: PubMed Central - PubMed

Affiliation: Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore Department of Physics, National University of Singapore, Singapore 117542, Singapore.

Show MeSH
Related in: MedlinePlus