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Holliday junction dynamics and branch migration: single-molecule analysis.

ABSTRACT: The Holliday junction (HJ) is a central intermediate in various genetic processes including homologous and site-specific recombination and DNA replication. Branch migration allows the exchange between homologous DNA regions, but the detailed mechanism for this key step of DNA recombination is unidentified. Here, we report direct real-time detection of branch migration in individual molecules. Using appropriately designed HJ constructs we were able to follow junction branch migration at the single-molecule level. Branch migration is detected as a stepwise random process with the overall kinetics dependent on Mg2+ concentration. We developed a theoretical approach to analyze the mechanism of HJ branch migration. The data show steps in which the junction flips between conformations favorable to branch migration and conformations unfavorable to it. In the favorable conformation (the extended HJ geometry), the branch can migrate over several base pairs detected, usually as a single large step. Mg2+ cations stabilize folded conformations and stall branch migration for a period considerably longer than the hopping step. The conformational flip and the variable base pair hopping step provide insights into the regulatory mechanism of genetic processes involving HJs.

SUBMITTER: Karymov M

PROVIDER: S-EPMC1140338 | biostudies-literature | 2005 Jun

REPOSITORIES: biostudies-literature

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Holliday junction dynamics and branch migration: single-molecule analysis.

Karymov Mikhail M Daniel Douglas D Sankey Otto F OF Lyubchenko Yuri L YL

Proceedings of the National Academy of Sciences of the United States of America 20050525 23

The Holliday junction (HJ) is a central intermediate in various genetic processes including homologous and site-specific recombination and DNA replication. Branch migration allows the exchange between homologous DNA regions, but the detailed mechanism for this key step of DNA recombination is unidentified. Here, we report direct real-time detection of branch migration in individual molecules. Using appropriately designed HJ constructs we were able to follow junction branch migration at the singl ...[more]

PMID: 15917329

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Project description: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.

Project description:The Holliday junction (HJ), or four-way junction, is a central intermediate state of DNA for homologous genetic recombination and other genetic processes such as replication and repair. Branch migration is the process by which the exchange of homologous DNA regions occurs, and it can be spontaneous or driven by proteins. Unfolding of the HJ is required for branch migration. 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. Folding of the HJ in one of the folded conformations terminates the branch migration phase. At the same time, in the unfolded state HJ rapidly migrates over entire homology region of the HJ in one hop. This process can be affected by irregularities in the DNA double helical structure, so mismatches almost terminate a spontaneous branch migration. Single-stranded breaks or nicks are the most ubiquitous defects in the DNA helix; however, to date, their effect on the HJ branch migration has not been studied. In addition, although nicked HJs are specific substrates for a number of enzymes involved in DNA recombination and repair, the role of this substrate specificity remains unclear. Our main goal in this work was to study the effect of nicks on the efficiency of HJ branch migration and the dynamics of the HJ. To accomplish this goal, we applied two single-molecule methods: atomic force microscopy and fluorescence resonance energy transfer. The atomic force microscopy data show that the nick does not prevent branch migration, but it does decrease the probability that the HJ will pass the DNA lesion. The single-molecule fluorescence resonance energy transfer approaches were instrumental in detailing the effects of nicks. These studies reveal a dramatic change of the HJ dynamics. The nick changes the structure and conformational dynamics of the junctions, leading to conformations with geometries that are different from those for the intact HJ. On the basis of these data, we propose a model of branch migration in which the propensity of the junction to unfold decreases the lifetimes of folded states, thereby increasing the frequency of junction fluctuations between the folded states.

Dataset Information

Holliday junction dynamics and branch migration: single-molecule analysis.

Publications

Holliday junction dynamics and branch migration: single-molecule analysis.

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