6] Topological structure of DNA knots and catenanes

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Publisher Summary This chapter discusses the basic concepts and methods by which the topological structure of DNA molecules is determined. Knotting and catenation are the true topological properties of DNA molecules. They can only be changed by breakage and reunion of the phosphodiester backbone of DNA, and not by any kind of structural deformation such as condensation or twisting. The two principal methods used for determining the topology of knots and catenanes are agarose gel electrophoresis and electron microscopy. Although only electron microscopy gives the complete stereostructure of individual molecules, electrophoresis provides a more rapid and quantitative overview of the population. It is best, therefore, to use both methods in combination. Gel electrophoresis can resolve even complex mixtures of nicked knots or catenanes into discrete ladders. Traditional techniques for the electron microscopy of DNA can be useful for identifying molecules as being knotted or catenated, but they provide insufficient visual information to score reliably the sign of the nodes. The complete topological characterization of knots and catenanes requires the introduction of the RecA protein coating method. The cooperative binding of E. coli RecA protein to single- or double-stranded DNA yields a thick complex that, when stained and shadowed, allows a reliable identification of DNA overlay and underlay at each crossing point.