Holliday junction dynamics and branch migration: Single-molecule analysis

Mikhail Karymov, Douglas Daniel, Otto F. Sankey, Yuri L Lyubchenko

Research output: Contribution to journalArticle

40 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)8186-8191
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume102
Issue number23
DOIs
StatePublished - Jun 7 2005

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Cruciform DNA
Genetic Phenomena
Base Pairing
Genetic Recombination
DNA
DNA Replication
Cations

Keywords

  • FRET
  • Fluorescence microscopy
  • Four-way junctions
  • Recombination DNA

ASJC Scopus subject areas

  • General

Cite this

Holliday junction dynamics and branch migration : Single-molecule analysis. / Karymov, Mikhail; Daniel, Douglas; Sankey, Otto F.; Lyubchenko, Yuri L.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 102, No. 23, 07.06.2005, p. 8186-8191.

Research output: Contribution to journalArticle

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N2 - 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.

AB - 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.

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