| Abstract: | The four-way DNA (Holliday) junction is an important postulated intermediate in the process of genetic recombination. Earlier studies have suggested that the junction exists in two alternative conformations, depending upon the salt concentration present. At high salt concentrations the junction folds into a stacked X structure, while at low salt concentrations the data indicate an extended unstacked conformation. The stereochemical conformation of the four-way DNA junction at low salt (low alkali ion concentration and no alkaline earth ions) was established by comparing the efficiency of fluorescence resonance energy transfer (FRET) between donor and acceptor molecules attached pairwise in three permutations to the 5' termini of the duplex arms. A new variation of FRET was implemented based upon a systematic variation of the fraction of donor labeled single strands. The FRET results indicate that the structure of the four-way DNA junction at low salt exists as an unstacked, extended, square arrangement of the four duplex arms. The donor titration measurements made in the presence of magnesium ions clearly show the folding of the junction into the X stacked structure. In addition, the FRET efficiency can be measured. The fluorescence anisotropy of the acceptor in the presence of Mg2+ during donor titrations was also measured; the FRET efficiency can be calculated from the anisotropy data and the results are consistent with the folded, stacked X structure. |
