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Published online on April 11, 2005, 10.1073/pnas.0409328102
PNAS | April 19, 2005 | vol. 102 | no. 16 | 5715-5720
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BIOPHYSICS
Observing spontaneous branch migration of Holliday junctions one step at a time

Sean A. McKinney *, Alasdair D. J. Freeman {dagger}, David M. J. Lilley {dagger}, and Taekjip Ha *, {ddagger}

*Department of Physics, University of Illinois at Urbana-Champaign, Urbana, IL 61801; and {dagger}Cancer Research UK Nucleic Acid Research Structure Group, Department of Biochemistry, University of Dundee, Dundee DD1 5EH, United Kingdom

Edited by Kiyoshi Mizuuchi, National Institutes of Health, Bethesda, MD, and approved March 2, 2005 (received for review December 14, 2004)

Genetic recombination occurs between homologous DNA molecules via a four-way (Holliday) junction intermediate. This ancient and ubiquitous process is important for the repair of double-stranded breaks, the restart of stalled replication forks, and the creation of genetic diversity. Once formed, the four-way junction alone can undergo the stepwise exchange of base pairs known as spontaneous branch migration. Conventional ensemble assays, useful for finding average migration rates over long sequences, have been unable to examine the affect of sequence and structure on the migration process. Here, we present a single-molecule spontaneous branch migration assay with single-base pair resolution in a study of individual DNA junctions that can undergo one step of migration. Junctions exhibit markedly different dynamics of exchange between stacking conformers depending on the point of strand exchange, allowing the moment at which branch migration occurs to be detected. The free energy landscape of spontaneous branch migration is found to be highly nonuniform and governed by two types of sequence-dependent barriers, with unmediated local migration being up to 10 times more rapid than the previously deduced average rate.

FRET | single molecule | recombination | DNA structure

Author contributions: S.A.M., D.M.L., and T.H. designed research; S.A.M. and A.D.J.F. performed research; A.D.J.F. contributed new reagents/analytic tools; S.A.M. and A.D.J.F. analyzed data; and S.A.M., D.M.L., and T.H. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Freely available online through the PNAS open access option.

{ddagger} To whom correspondence should be addressed. E-mail: tjha{at}uiuc.edu.

© 2005 by The National Academy of Sciences of the USA


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