THE SOLUTION STRUCTURE OF THE 4-WAY DNA JUNCTION AT LOW-SALT CONDITIONS - A FLUORESCENCE RESONANCE ENERGY-TRANSFER ANALYSIS

The four-way DNA (Holliday) junction is an important postulated interm ediate in the process of genetic recombination. Earlier studies have s uggested that the junction exists in two alternative conformations, de pending upon the salt concentration present. At high salt concentratio ns the junction folds into a stacked X structure, while at low salt co ncentrations 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 establis hed by comparing the efficiency of fluorescence resonance energy trans fer (FRET) between donor and acceptor molecules attached pairwise in t hree permutations to the 5' termini of the duplex arms. A new variatio n of FRET was implemented based upon a systematic variation of the fra ction of donor labeled single strands. The FRET results indicate that the structure of the four-way DNA junction at low salt exists as an un stacked, extended, square arrangement of the four duplex arms. The don or titration measurements made in the presence of magnesium ions clear ly show the folding of the junction into the X stacked structure. In a ddition, the FRET efficiency can be measured. The fluorescence anisotr opy of the acceptor in the presence of Mg2+ during donor titrations wa s also measured; the FRET efficiency can be calculated from the anisot ropy data and the results are consistent with the folded, stacked X st ructure.