Findings from University of Nebraska broaden understanding of DNA research
December 22nd, 2008
2008 DEC 22 -- "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," researchers in the United States report.
"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," wrote M.A. Karymov and colleagues, University of Nebraska.
The researchers concluded: "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."
Karymov and colleagues published their study in Biophysical Journal (Structure, Dynamics, and Branch Migration of a DNA Holliday Junction: A Single-Molecule Fluorescence and Modeling Study. Biophysical Journal, 2008;95(9):4372-4383).
For additional information, contact Y.L. Lyubchenko, University of Nebraska, Medical Center, Dept. of Pharmaceutical Science, 986025 Nebraska Med Center, Omaha, NE 68198, USA.
Publisher contact information for the Biophysical Journal is: Biophysical Society, 9650 Rockville Pike, Bethesda, MD 20814-3998, USA.
Keywords: United States, Omaha, DNA, DNA Replication, DNA Research, Genetics, Medical Device, University of Nebraska.
This article was prepared by Proteomics Weekly editors from staff and other reports. Copyright 2008, Proteomics Weekly via NewsRx.com.
