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Recombination of genes is essential to the evolution of genetic diversity, the segregation of chromosomes during cell division, and certain DNA repair processes. The Holliday junction, a four-arm, four-strand branched DNA crossover structure, is formed as a transient intermediate during genetic recombination and repair processes in the cell. The recognition and subsequent resolution of Holliday junctions into parental or recombined products appear to be critically dependent on their three-dimensional structure. Complementary NMR and time-resolved f luorescence resonance energy transfer experiments on immobilized four-arm DNA junctions reported here indicate that the Holliday junction cannot be viewed as a static structure but rather as an equilibrium mixture of two conformational isomers. Furthermore, the distribution between the two possible crossover isomers was found to depend on the sequence in a manner that was not anticipated on the basis of previous low-resolution experiments.Genetic recombination is a primary biological process that is critical to the continual evolution of all organisms, and the Holliday junction is a requisite intermediate in nearly all recombination events. Holliday (1) proposed the first and most basic mechanism of genetic recombination: homologous chromatids are nicked at identical sites, then the duplex DNA is partially unwound and the single strands are paired with the complementary strands of the homologs. The structure thus formed, termed the Holliday junction (HJ), may travel (branch-migrate) in either direction along the DNA. Eventually, the structure is cut endonucleolytically (resolved) to give two chromosomes with either a parental (patch) or a recombined (splice) configuration (Fig. 1). Although many postulated mechanisms for recombination exist, they differ only in how recombination is initiated and in how DNA strands are displaced (1-5). Most recombination mechanisms invoke formation of heteroduplex DNA molecules containing branched structures.It has been reported for more than 40 years that DNA sequence at or near a HJ influences how it is resolved and the corresponding ratio of parental to recombined molecules (6).Results from a variety of experiments suggest that resolution must be predetermined for certain sequences and that not all HJs are identical in structure (e.g., refs. 7-10). To gain an in-depth understanding of recognition and resolution of HJs, it is imperative to characterize their structure and dynamics at the atomic level and to establish how these properties vary with sequence.A consensus view of HJ structure has emerged from biophysical studies of immobilized four-arm DNA junctions in which the sequence is designed to eliminate the possibility of branch migration (for reviews see refs. 11 and 12). The four helical arms are seen to stack upon each other in a pairwise fashion, forming two duplex domains with an acute interhelical angle (Fig. 2). In this configuration, two contiguous strands run antiparallel to each other and the two crossover stra...

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