The four-way DNA (Holliday) junction is an essential intermediate in DNA recombination, and its dynamic characteristics are likely to be important in its cellular processing. In our previous study we observed transitions between two antiparallel stacked conformations using a single-molecule fluorescence approach. The magnesium concentration-dependent rates of transitions between stacking conformers suggested that an unstacked open structure, which is stable in the absence of metal ions, is an intermediate. Here, we sought to detect possible rare species such as open and parallel conformations and further characterized ionic effects. The hypothesized open intermediate cannot be resolved directly due to the limited time resolution and sensitivity, but our study suggests that the open form is achieved very frequently, hundreds of times per second under physiologically relevant conditions. Therefore despite being a minority species, its frequent formation raises the probability that it could become stabilized by protein binding. By contrast, we cannot detect even a transient existence of the junctions in a parallel form, and the probability of such forms with a lifetime greater than 5 ms is less than 0.01%. Stacking conformer transitions are observable in the presence of sodium or hexammine cobalt (III) ions as well as magnesium ions, but the transition rates are higher for lower valence ions at the same concentrations. This further supports the notion that electrostatic stabilization of the stacked structures dictates the interconversion rates between different structural forms.
Bibliographical noteFunding Information:
We thank A.-C. Déclais for providing junction 7; I. Rasnik, S. Myong, S. Hohng and R. Roy for advice and discussion on the experiment. C.J. thanks J. Milton for his guidance. Funding was provided by the NIH, NSF, Searle Scholars Award (T.H.) and by Cancer Research-UK (D.M.J.L.). S.A.M. was partially supported by the NIH molecular biophysics training grant and the NSF graduate fellowship.
- branch migration
- FRET, fluorescence resonance energy transfer
- Holliday junction
- ionic effect
- parallel conformation
- single molecule spectroscopy