Structure, Dynamics, and Branch Migration of a DNA Holliday Junction: A Single-Molecule Fluorescence and Modeling Study

Abstract

The Holliday junction (HJ) is a central intermediate of various genetic processes, including homologous and site-specific DNA recombination and DNA replication. Elucidating the structure and dynamics of HJs provides the basis for understanding the molecular mechanisms of these genetic processes. Our previous single-molecule fluorescence studies led to a model according to which branch migration is a stepwise process consisting of consecutive migration and folding steps. These data led us to the conclusion that one hop can be more than 1 basepair (bp); moreover, we hypothesized that continuous runs over the entire sequence homology (5 bp) can occur. Direct measurements of the dependence of the fluorescence resonance energy transfer (FRET) value on the donor-acceptor (D-A) distance are required to justify this model and are the major goal of this article. To accomplish this goal, we performed single-molecule FRET experiments with a set of six immobile HJ molecules with varying numbers of bps between fluorescent dyes placed on opposite arms. The designs were made in such a way that the distances between the donor and acceptor were equal to the distances between the dyes formed upon 1-bp migration hops of a HJ having 10-bp homology. Using these designs, we confirmed our previous hypothesis that the migration of the junction can be measured with bp accuracy. Moreover, the FRET values determined for each acceptor-donor separation corresponded very well to the values for the steps on the FRET time trajectories, suggesting that each step corresponds to the migration of the branch at a defined depth. We used the dependence of the FRET value on the D-A distance to measure directly the size for each step on the FRET time trajectories. These data showed that one hop is not necessarily 1 bp. The junction is able to migrate over several bps, detected as one hop and confirming our model. The D-A distances extracted from the FRET properties of the immobile junctions formed the basis for modeling the HJ structures. The composite data fit a partially opened, side-by-side model with adjacent double-helical arms slightly kinked at the four-way junction and the junction as a whole adopting a global X-shaped form that mimics the coaxially stacked-X structure implicated in previous solution studies.

Single-Molecule Dynamics and Mechanisms of Metalloregulators and Metallochaperones

“…In the presence of Na + and Mg 2+ , it folds into two X-shaped stacked conformers (conf-I and conf-II, Figure 5E), where each conformer could be viewed approximately as two B-form helices forming a cross structurally (46, 47). The two conformers interconvert dynamically at room temperature (Figure 5E), and the structural dynamics of a single HJ molecule can be followed in real time by smFRET measurement where two of the HJ's four arms are labeled with a FRET pair (45, 48–50). We engineered a HJ and encoded in its arms the dyad symmetric sequence recognized by CueR (Figure 5E).…”

Hexapeptides That Inhibit Processing of Branched DNA Structures Induce a Dynamic Ensemble of Holliday Junction Conformations

“…Nevertheless, the peak FRET values are distinct from those observed under the low ionic strength conditions that favor the square planar conformations, indicating a set of distinct conformations. Although the canonical model involving stacked X and square planar conformations has provided an important foundation for understanding junction conformations and dynamics, there is increasing evidence for additional, noncanonical conformations along the pathways for exchange between stacked X conformations (19,20), as stable ground states for mobile junctions (22), or in complexes with junctionbinding proteins (21). It is possible that the peptide-induced conformational ensembles observed here resemble one or more of these noncanonical junction conformations.…”

Effect of Single-Strand Break on Branch Migration and Folding Dynamics of Holliday Junctions

“…Compared with this pause, the branch migration phase is fast (~100 ms per hop). The availability of FRET data for a set of HJs with different donor-acceptor distances allowed us to refine the models of immobile HJs (12). The composite data fit a partially opened, side-by-side model with adjacent double-helical arms slightly kinked at the four-way junction, and the junction as a whole adopts a global X-shaped form that mimics the coaxially stacked-X structure implicated in previous solution studies.…”

“…The original time trajectories of fluorescence intensity were acquired as described previously using the same confocal setup (11,12,21,23). A thoroughly cleaned sample cell formed by a sandwiched glass slide, Teflon spacer ring, and quartz coverslip was filled with 1 mg/mL biotinylated BSA (Sigma, St. Louis, MO) solution in pH 7.5 TES buffer (10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA) for 10 min, rinsed three times with the same TES buffer, then filled with 0.2 mg/mL solution of streptavidin (Sigma) in the same buffer for 10 min, and again rinsed three times with TES buffer.…”