RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations - PubMed (original) (raw)
RecA binding to a single double-stranded DNA molecule: a possible role of DNA conformational fluctuations
J F Leger et al. Proc Natl Acad Sci U S A. 1998.
Abstract
Most genetic regulatory mechanisms involve protein-DNA interactions. In these processes, the classical Watson-Crick DNA structure sometimes is distorted severely, which in turn enables the precise recognition of the specific sites by the protein. Despite its key importance, very little is known about such deformation processes. To address this general question, we have studied a model system, namely, RecA binding to double-stranded DNA. Results from micromanipulation experiments indicate that RecA binds strongly to stretched DNA; based on this observation, we propose that spontaneous thermal stretching fluctuations may play a role in the binding of RecA to DNA. This has fundamental implications for the protein-DNA binding mechanism, which must therefore rely in part on a combination of flexibility and thermal fluctuations of the DNA structure. We also show that this mechanism is sequence sensitive. Theoretical simulations support this interpretation of our experimental results, and it is argued that this is of broad relevance to DNA-protein interactions.
Figures
Figure 1
(A) Force vs. extension for λ-EMBL3, both before (solid line) and after (dashed line) complete RecA polymerization, showing elongation of the dsDNA. The difference in low-force behaviors (<10 pN) of the two curves shows that the nucleofilament has a larger persistence length than the initial dsDNA molecule. In the case of the nucleofilament, such force–extension curves are independent of the force applied during RecA polymerization. (B) Time dependence of elongation of a single λ-EMBL3 during RecAbinding for various fixed forces in presence of 1 mM ATP-γS. The initial time dependence of the elongation is highly reproducible, but the late time behavior (i.e., beyond 80% of the full elongation caused by RecA binding) is much more variable from one experiment to another. This might be because of nicks that, as RecA binds, become covered, making progressively more of the dsDNA twist-blocked and thereby limiting the rate of the final stages of polymerization.
Figure 2
Probability distribution P(u) for local stretching, u, of a dsDNA molecule for various applied forces. The bimodal behavior observed for 55 pN is caused by the coexistence of B-DNA and S-DNA on the transition plateau (see force–extension data in Fig. 1_A_). The asymmetry in P(u) for 75 pN arises because of the steric limit, u ≈ 0.9, where the dsDNA phosphodiester backbones are stretched fully. Inset shows P(u) close to u = 0.5.
Figure 3
Time dependence of elongation of a dsDNA molecule obtained from an Ising-type model for various applied forces.
Figure 4
(A) Force vs. extension data for λ-EMBL3 (solid line) and 156Gmac (dotted line). These two curves were obtained from a single preparation by using a single fiber, thus avoiding any complication of comparison caused by slight changes in calibration of fiber displacement and/or stiffness. Two sets of beads (each one carrying either λ-EMBL3 or 156Gmac) were injected in the cuvette. Thus, two different types of beads became grafted to the fiber via either λ-EMBL3 or 156Gmac. Force–extension diagrams shown here result from successive experiments performed on the two different sets of beads. Extensions are given as fractions of B-form fully extended length to allow comparison of the two different molecules. A fit to the extensible worm-like-chain model (21) gives the persistence length A and the stretching modulus γ for the two molecules. For 156Gmac, A = 55 nm and γ = 270 nm−1; for λ-EMBL3, A = 75 nm and γ = 450 nm−1. The transition to S-DNA starts at 50 pN for 156Gmac instead of 55 pN for λ-EMBL3, and the transition width (in terms of force) is larger for 156Gmac than for λ-EMBL3. Finally, the overstretching of 156Gmac (u = 0.75) is larger than the overstretching of λ-EMBL3 (u = 0.65). According to these data, 156Gmac should exhibit stronger local structural thermal fluctuations than λ- EMBL3. (B) Comparison of RecA binding kinetics to λ-EMBL3, λ-EMBL3 dimer, and 156Gmac for a constant externally applied force of 45 pN. Extension is plotted as a fraction of B-form length to allow comparison of the three different molecules. Comparison of λ-EMBL3 and λ-EMBL3 dimer shows that the faster RecA binding in the case of 156Gmac is caused by the differing base content and is not an artifact of differing molecule lengths.
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