Structure and dynamics of three-way DNA junctions: atomic force microscopystudies

We have used atomic force microscopy (AFM) to study the conformation of thr ee-way DNA junctions, intermediates of DNA replication and recombination, I mmobile three-way junctions with one hairpin arm (50, 27, 18 and 7 bp long) and two relatively long linear arms were obtained by annealing two partial ly homologous restriction fragments. Fragments containing inverted repeats of specific length formed hairpins after denaturation, Three-way junctions were Obtained by annealing one strand of a fragment from:a parental plasmid with one strand of an inverted repeat-containing fragment, purified from g els, and examined by AFM, The molecules are clearly seen as three-armed mol ecules with one short aim and two flexible long arms, The AFM analysis reve aled two important features of three-way DNA junctions, First, three-way ju nctions are very dynamic structures. This conclusion is supported by a high variability of the inter-arm angle detected on dried samples. The mobility of the junctions was observed directly by imaging the samples in liquid (A FM;in situ), Second, measurements of the angle between the arms led to the conclusion that three-way junctions are not flat, but rather pyramid-like, Non-flatness of the junction should be taken into account in analysis of th e AFM data.