Observing spontaneous branch migration of Holliday junctions one step at a time

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   Fig. 1.

   Dynamics of structural interconversion in a junction capable of a single step of branch migration. (A) Structural interconversion possible in junction J₁TGGG. The junction comprises four arms (B, H, R, and X) formed by annealing four ssDNA molecules of 22 nt each (named b, h, r, and x). The sequence allows one step of branch migration (vertical interconversion). In one species (U, Upper) the four arms are each 11 bp in length, whereas in the other form (M, Lower) the arms are 12 bp (B and R) or 10 bp (H and X). Each of these species can exist in either stacking conformer (horizontal interconversion) isoI (H on B stacking) or isoII (B on X stacking). Depicted intermediate species are not observed directly in our experiments. Other sequences varied either the migrating base pair (J₁TCGC, J₁TAGA, J₁TTGT) or the base pair flanking the migrating base pair in the X arm (J₁TGGGt, J₁TGGGa) (see Materials and Methods for naming scheme). In all cases, the Cy3 donor, Cy5 acceptor, and biotin were attached to the 5′ termini of the h, b, and r strands, respectively. (B) The central sequences of the full set of junctions analyzed in these studies, with the sequence differences from J₁TGGG highlighted in magenta. Lines indicate the base pairs that form in the alternative branch position.

   
   
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   Fig. 2.

   Fluorescence time traces for single junction molecules. Solution conditions are as described in the text with a Mg²⁺ concentration of 50 mM. (A) A record of donor (green) and acceptor (red) fluorescence intensities (Upper) and calculated FRET efficiency (Lower) from a single molecule of J₁TGGGa as a function of time. The molecule interconverts between high and low FRET efficiency and exhibits two distinct dynamic behaviors. Arrows denote a transition between these forms at 3 s and the reverse at 7 s. (B) FRET efficiency as a function of time for J₁TGGG. (C) FRET efficiency as a function of time for J₁TTGT.

   
   
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   Fig. 3.

   Determination of the major conformational species in solution by hydroxyl radical probing. Four junctions were constructed by hybridization of four strands, one of which was radioactively 5′-³²P-labeled. The same strand was also hybridized to its complement to make a perfect duplex for comparison. The junction and duplex species were subjected to hydroxyl radical attack, and the products were analyzed by gel electrophoresis under denaturing conditions and phosphorimaging. The radioactivity in each band was quantified from the phosphorimage files and presented in plotted form for the duplex (gray) and junction (red). Regions protected against radical attack are revealed by the lower intensity of the junction-derived peaks. (A) The profiles of radical attack for the four strands of junction J₁TGGG. There are regions of protection apparent at the centers of the b and r strands (labeled with the corresponding nucleotides). (Insets) The ratios of intensities are also shown by the histograms, clearly showing the protection of the central nucleotides. The results show clearly that the major species in solution is in the isoII stacking conformation (where the exchanging b and r strands are protected) and has the U branch position (shown by the position of protection on these strands). The protected nucleotides are summarized in C.(B) Comparison of the radical attack profiles on the b strands of junctions J₁TGGGt, J₁TTGT, and J₁TCGC. The positions of protection indicate that J1TGGGt is also in the U, isoII conformation, whereas J₁TTGT is predominantly in the alternative branch position, i.e., M, isoII. By contrast, the protection of J₁TCGC indicates that all of the stacking and branch positions are populated in this junction. The full data are shown in Figs. 9–12, which are published as supporting information on the PNAS web site, and the protected nucleotides are summarized in C. (C) Summary of the nucleotides protected against radical attack in the four junctions, shown by the yellow shading. The nucleotides highlighted in red can participate in branch migration. Junction J₁TAGA (Fig. 12) is very similar to J₁TCGC.

   
   
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   Fig. 4.

   Branch migration energy landscape. (A) The number of conformational transitions occurring before a migration step takes place (α) is approximately constant for G-C base pairs, but the value is halved for an A-T base pair. Empty bars represent the α value for slow-to-fast state transitions, and filled bars represent the α value for fast-to-slow transitions. (B) Arrhenius plot showing the dependence of the rate of branch migration on temperature. Note that the activation enthalpy for branch migration requiring the breakage of a G-C base pair (J₁TGGG) is nearly three times that for an A-T pair (J₁TTGT). (C) Illustrative scheme showing the important states and energy barriers for a single-step junction. Although open states are depicted, they are too short-lived for direct experimental observation in the presence of Mg²⁺ ions. Note that the open state is a common intermediate for both conformer exchange and branch migration. (D) Long-range branch migration will be a very uneven process, reflecting a rugged energy landscape. The data from junctions of different sequence indicate that the lifetimes of individual stacking conformer lifetimes vary markedly, thereby trapping junctions in particularly stable step positions while moving relatively rapidly through stretches of less stable positions.