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Figure 1 Experimental design. (A) Holliday junctions are preformed by annealing of two branch migration substrates, S1* and S2. Branch migration to the distal end results in irreversible dissociation of the Holliday junction intermediate and formation of two heteroduplex products (P1* and P2). (B) pBR322-derived Holliday junctions used in this study (not to scale). The boxed gray regions represent heterologous sequences. The arrow indicates the location of the 3-bp mutation in the vicinity of the AvaI site. The asterisk denotes a 32P-label.
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 | Figure 2 Population-averaged branch migration assay. (A) Branch migration of HJ 20/0 at 37 and 20°C. The first lane in both gels contained 32P-labeled S1 in the absence of S2. (B) Restriction map of S1* and P1*. AvaI and EcoRV restriction sites are located at 50 and 22 bp, respectively. (C) Control of the ratio between S1* and P1* generated in 900 s of branch migration at 37°C (left panel) and 20°C (right panel). S1* (lanes 1–3, 7 and 8) was digested with AvaI (lanes 2 and 8) or with EcoRV (lane 3). Branch migration products obtained after 900 s of incubation at 37°C (lanes 4–6) or 20°C (lanes 9 and 10) were digested with AvaI (lanes 5 and 10) or EcoRV (lane 6), electrophoresed on 6% polyacrylamide gels and quantified. AI, AvaI; RV, EcoRV.
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Figure 3 Computer simulations of RuvAB-directed branch migration. Simulations are shown for experiments performed in the absence (A) or in the presence (B) of a medially located sequence heterology. The different parameters used for computer simulations are shown (see text for details).
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 | Figure 4 Kinetic modeling of branch migration. Results are shown for experiments carried out at 37°C (A) and 20°C (B). Filled circles, experimental points of monomer-product appearance during branch migration assays; lines, computer-generated simulations. Branch migration is expressed as the fraction of the monomer band relative to the total amount of radioactivity for each time point. (C) Minimization analysis for the 'best fit' translocation rate for branch migration at 20°C. Deviation is the normalized  2 (see Materials and methods).
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Figure 5 RuvAB-directed branch migration is impeded by sequence heterology. (A) Time course of monomer-product appearance during branch migration of HJ 20/0 (filled circles). The lines represent computer-generated simulations of the appearance of S1*+P1* during branch migration. For comparison, the simulations of branch migration generated with  het=20 and 200 s are also shown. (B) Branch migration in the presence of a medially located 40-bp heterology. Open circles, HJ 40/0; open triangles, HJ 40/40. In (A, B), the ordinate axis is the same as in Figure 4. (C, D) Minimization analysis for the 'best fit' bypass time (het) through 20-bp (filled circles) or 40-bp (open circles) heterologies and for the 'best fit' lifetime (life) of the complex stalled at 20- or 40-bp heterologies, respectively.
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 | Figure 6 'Single-molecule' branch migration assays. (A) A Holliday junction is attached to the glass surface via its long arms. RuvAB loading on the short arms of the junction results in a shortening of the tether, leading to a decrease in the amplitude of bead Brownian motion. (B) The 2- and 3-kb Holliday junctions used for TPM. The Holliday junctions were modified with digoxygenin (solid circles) and biotin (solid squares) at the 5' ends of the long duplex arms. (C) Positions of the bead (given in pixels), tethered with a 2-kb Holliday junction, after the addition of RuvAB and ATP as a function of time. (D) Time course of Brownian motion of the bead in (C) calculated at 4-s intervals. (E) Five examples of time courses of RuvAB-directed branch migration of 2-kb (filled circles) and 3-kb Holliday junctions (filled triangles). The numbers shown correspond to the observed rates of branch migration determined from the slopes of the linear fits (bp/s). (F) Distribution of individual branch migration rates (bin width 8 bp/s).
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Figure 7 RuvAB-directed branch migration of individual Holliday junctions in the presence of a medially located 40-bp sequence heterology. (A) The 2-kb Holliday junction containing a 40-bp heterologous insert (2-kb HJ 40/40). (B) Histograms for several hundred amplitudes of Brownian motion before (filled circles) and after (open circles) branch migration collected from 30 individual branch migrations. The lines correspond to best fits for a Gaussian distribution with 143 33 and 11219 nm (means.d.) for each population, respectively. (C) Individual branch migration time course showing oscillations between forward and backward translocations of a Holliday junction constrained between two 40-bp blocks of heterology. (D) Bypass through a medially located 40-bp heterology.
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