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Nucleic Acids Research, 2000, Vol. 28, No. 18 3472-3477
© 2000 Oxford University Press

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

Luda S. Shlyakhtenko1, Vladimir N. Potaman3, Richard R. Sinden3, Alexander A. Gall4 and Yuri L. Lyubchenko1,2,*

1Department of Microbiology and 2Department of Biology, Arizona State University, Tempe, AZ 85287-2701, USA, 3Institute of Biosciences and Technology, Texas A&M University, 2121 West Holcombe Boulevard, Houston, TX 77030-3303, USA and 4Epoch Biosciences Inc., 12277 134th Court NE, Number 110, Redmond, WA 98052, USA

We have used atomic force microscopy (AFM) to study the conformation of three-way DNA junctions, intermediates of DNA replication and recombination. Immobile 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 partially 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 gels, and examined by AFM. The molecules are clearly seen as three-armed molecules with one short arm and two flexible long arms. The AFM analysis revealed two important features of three-way DNA junctions. First, three-way junctions 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 (AFM 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 the AFM data.

* To whom correspondence should be addressed at: Department of Microbiology, Arizona State University, PO Box 872701, Tempe, AZ 85287-2701, USA. Tel: +1 480 965 8430; Fax: +1 480 965 0098; Email: yuri.lyubchenko@asu.edu


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