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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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De-polymerization of preformed MtRecA filaments at different MtRecX concentrations. (A). Time traces of the extension obtained on three independent preformed MtRecA filaments formed on three ssDNA tethers (indicated by different colors) after addition of 80 nM MtRecX at forces of 2 - 4 pN. Inset shows steps in zoom-in time traces. (B) and (C) show extension time traces of three independent preformed MtRecA filaments at 400 nM MtRecX (B) and 1 μM MtRecX (C) at forces of 2–4 pN. The red lines in (A)–(C) show stepwise de-polymerization and re-polymerization obtained from a step finding algorithm (Method S3). (D) A long extension time trace of a preformed MtRecA in 1 μM MtRecX, 1 μM MtRecA, 1 mM ATPγS (other conditions remained the same), where no net de-polymerization of MtRecA filament occurs over the experimental time scale.
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Figure 2: De-polymerization of preformed MtRecA filaments at different MtRecX concentrations. (A). Time traces of the extension obtained on three independent preformed MtRecA filaments formed on three ssDNA tethers (indicated by different colors) after addition of 80 nM MtRecX at forces of 2 - 4 pN. Inset shows steps in zoom-in time traces. (B) and (C) show extension time traces of three independent preformed MtRecA filaments at 400 nM MtRecX (B) and 1 μM MtRecX (C) at forces of 2–4 pN. The red lines in (A)–(C) show stepwise de-polymerization and re-polymerization obtained from a step finding algorithm (Method S3). (D) A long extension time trace of a preformed MtRecA in 1 μM MtRecX, 1 μM MtRecA, 1 mM ATPγS (other conditions remained the same), where no net de-polymerization of MtRecA filament occurs over the experimental time scale.

Mentions: To determine how a preformed MtRecA filament is regulated when MtRecX is added to the MtRecA reaction solution, we investigated the effects of MtRecX on the dynamics of disassembly of preformed MtRecA filaments. At low concentrations of MtRecX (80 nM), we observed progressive reductions in DNA extension at ∼3 pN (Figure 2A), indicating net MtRecA filament disassembly. Although noisy, the disassembly process appeared stepwise overall. In addition to the dominant disassembly process, occasionally large step (> 10 nm) MtRecA re-assembly events were also observed (arrows, Figure 2A).


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)

De-polymerization of preformed MtRecA filaments at different MtRecX concentrations. (A). Time traces of the extension obtained on three independent preformed MtRecA filaments formed on three ssDNA tethers (indicated by different colors) after addition of 80 nM MtRecX at forces of 2 - 4 pN. Inset shows steps in zoom-in time traces. (B) and (C) show extension time traces of three independent preformed MtRecA filaments at 400 nM MtRecX (B) and 1 μM MtRecX (C) at forces of 2–4 pN. The red lines in (A)–(C) show stepwise de-polymerization and re-polymerization obtained from a step finding algorithm (Method S3). (D) A long extension time trace of a preformed MtRecA in 1 μM MtRecX, 1 μM MtRecA, 1 mM ATPγS (other conditions remained the same), where no net de-polymerization of MtRecA filament occurs over the experimental time scale.
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Related In: Results  -  Collection

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Figure 2: De-polymerization of preformed MtRecA filaments at different MtRecX concentrations. (A). Time traces of the extension obtained on three independent preformed MtRecA filaments formed on three ssDNA tethers (indicated by different colors) after addition of 80 nM MtRecX at forces of 2 - 4 pN. Inset shows steps in zoom-in time traces. (B) and (C) show extension time traces of three independent preformed MtRecA filaments at 400 nM MtRecX (B) and 1 μM MtRecX (C) at forces of 2–4 pN. The red lines in (A)–(C) show stepwise de-polymerization and re-polymerization obtained from a step finding algorithm (Method S3). (D) A long extension time trace of a preformed MtRecA in 1 μM MtRecX, 1 μM MtRecA, 1 mM ATPγS (other conditions remained the same), where no net de-polymerization of MtRecA filament occurs over the experimental time scale.
Mentions: To determine how a preformed MtRecA filament is regulated when MtRecX is added to the MtRecA reaction solution, we investigated the effects of MtRecX on the dynamics of disassembly of preformed MtRecA filaments. At low concentrations of MtRecX (80 nM), we observed progressive reductions in DNA extension at ∼3 pN (Figure 2A), indicating net MtRecA filament disassembly. Although noisy, the disassembly process appeared stepwise overall. In addition to the dominant disassembly process, occasionally large step (> 10 nm) MtRecA re-assembly events were also observed (arrows, Figure 2A).

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