Yonatan Savir, Tsvi Tlusty  Molecular Cell 

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RecA-Mediated Homology Search as a Nearly Optimal Signal Detection System  Yonatan Savir, Tsvi Tlusty  Molecular Cell  Volume 40, Issue 3, Pages 388-396 (November 2010) DOI: 10.1016/j.molcel.2010.10.020 Copyright © 2010 Elsevier Inc. Terms and Conditions

Molecular Cell 2010 40, 388-396DOI: (10.1016/j.molcel.2010.10.020) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 Homologous Recombination Mediated by Bacterial RecA (A) RecA monomers polymerize along an ssDNA to form a nucleoprotein filament (NPF). (B) The ssDNA inside the NPF is deformed nonuniformly (PDB 3CMU [Chen et al., 2008]; RecA protein is not shown). Each nucleotide triplet has a B-DNA like structure. Most of the deformation is localized between the triplets such that the axial rise between neighboring triplets is increased from 3.4 Å to about 7.8 Å. (C) The NPF searches for homology along a dsDNA. The homology check involves short segments in a stepwise fashion. The dsDNA deforms nonuniformly in order to align its base pairs with those of the RecA-polymerized ssDNA and thus allows recognition via Watson-Crick pairing. Therefore, the triplets of the dsDNA are slightly deformed, but for the next triplet to align with the ssDNA bases, the dsDNA has to locally extend by a factor of almost 2.5 (from 3.4 Å to about 8 Å). The aligned base pairs can be complementary (green rectangles) or noncomplementary (green and red rectangles). Once the bases are aligned, a “decision” is made. If the segments are heterologous, the synapse is destabilized and may disintegrate. If they are homologous, the synapse is stable and the process may proceed by checking a subsequent segment for homology. (D) The paired DNA within the RecA filament exhibits three-base periodicity. Most of the deformation is localized between the slightly deformed triplets (PDB 3CMT [Chen et al., 2008]; RecA protein is not shown). (E) Homology check and strand exchange occur at one end of the synapse. At the other end, RecA depolymerizes and allows the displacement of the exchanged ssDNA. Molecular Cell 2010 40, 388-396DOI: (10.1016/j.molcel.2010.10.020) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Decision Table and Tree in Homologous Recombination The inputs are whether the RecA-polymerized ssDNA (NPF) encounters a complementary dsDNA segment (+) or a noncomplementary one (−). The outcomes are whether to proceed with HR (+) or to abort it (−). There are four input-output combinations: the correct true positive (+, +) and true negative (−, −), and the incorrect false positive (+, −), i.e., “false alarm,” and false negative (−, +), i.e., “miss.” pij is the probability of receiving an input, j, and making a decision, i. The weight, Cij, measures the impact of such a decision on the system. Other recognition processes can be described by a similar decision table. Molecular Cell 2010 40, 388-396DOI: (10.1016/j.molcel.2010.10.020) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 RecA-DNA Interaction Is Nearly Optimal for Sequence Detection (A) Top: As the total binding free energy per bp ΔGt increases, the binding probability to a complementary sequence, pb(+) (blue), increases faster than the binding probability to a noncomplementary one, pb(−) (red). Bottom: The detection cost C (Equation 2) is the weighted difference between these two binding curves (green). Minimizing the detection cost amounts to maximizing the binding probability to a complementary segment while minimizing the binding probability to a noncomplementary one. The optimal free energy is ΔGt¯=(m/2N)·ΔΔGmis (plotted for tolerance t = 1). (B) The detection cost C (Equation 2) for a recognition step 3 bp long, N = 3, as a function of ΔGt for t = 1. C was calculated for all possible competitors, of m = 1 (green), 2 (red), and 3 (blue) mismatches. The cost function C for each competitor appears as a band whose width is determined by the experimental range of the destabilization free energy ΔΔGmis = 1.5−3.5 kBT. The central value for each of the bands is denoted by a solid line. The shading denotes the experimental range of ΔGt = 0.5−1.5 kBT. For all competitors, the measured free energy is near the minimum of the cost curves and therefore enables nearly optimal detection. (C) The detection cost for various tolerance values, t: the symmetric case, t = 1 (blue); low error tolerance, t < 1 (red); high error tolerance, t > 1 (green); and extremely low error tolerance, t < tc = exp(–m·ΔΔGmis) (cyan, normalized by t). In this figure, m = 2, ΔΔGmis = 2.5 kBT, and N = 3. In the case of HR, a rough estimate of t is t ≈1 (Supplemental Experimental Procedures). At low error tolerance, t < 1, avoiding incorrect binding is of higher priority than binding correctly, and therefore low ΔGt values are more beneficial relative to the symmetric case. The opposite phenomenon occurs at high error tolerance (see the main text). For systems of extremely low error tolerance, the cost has a sharp sigmoidal shape with a threshold at ΔGt = (m/N)·ΔΔGmis. The largest binding energy that still resides in the lower part of the sigmoid is near the transition point. Therefore, even in at the extreme where the tolerance is very low, free energy values of about 1 kBT are advantageous (see the main text). Molecular Cell 2010 40, 388-396DOI: (10.1016/j.molcel.2010.10.020) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Binding and Extension Are Tuned. The binding probabilities, pb(+) (blue) and pb(−) (red), and the detection cost, C (green), as a function of the extension free energy per triplet, ΔGext. The recognition step is 3 bp long. As the extension increases, the binding probabilities decrease such that pb(+) decreases slower than pb(−). Thus, the value of extension free energy that balances the opposite trends and minimizes the detection cost is ΔGext¯=3(ΔGb¯−(m/6)·ΔΔGmis). As long as ΔGb > (m/6) · ΔΔGmis ≈1 kBT, the optimal extension is nonzero, i.e., RecA-mediated extension enhances sequence detection. Inset: The experimental (black lines) and the theoretical (blue band) limitations on ΔGext and ΔGb. The optimal values (blue band) (Equation 4) are calculated, for all possible competitors (m = 1, 2, 3) and the range of measured values of ΔΔGmis. The resulting bands are parallel and overlapping, a direct outcome of the optimality of ΔGt. Molecular Cell 2010 40, 388-396DOI: (10.1016/j.molcel.2010.10.020) Copyright © 2010 Elsevier Inc. Terms and Conditions