Journal Article

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

Luda S. Shlyakhtenko, Vladimir N. Potaman, Richard R. Sinden, Alexander A. Gall and Yuri L. Lyubchenko

in Nucleic Acids Research

Volume 28, issue 18, pages 3472-3477
Published in print September 2000 | ISSN: 0305-1048
Published online September 2000 | e-ISSN: 1362-4962 | DOI: http://dx.doi.org/10.1093/nar/28.18.3472
Structure and dynamics of three-way DNA junctions: atomic force microscopy studies

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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.

Journal Article.  4024 words.  Illustrated.

Subjects: Chemistry ; Biochemistry ; Bioinformatics and Computational Biology ; Genetics and Genomics ; Molecular and Cell Biology

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