HYDROGEN EXCHANGE STUDIES OF DNA STRUCTURE.

Abstract

DNA unwinding, and may well control the accessibility of the structure to nucleases and polymerases. In principle, hydrogen exchange methods afford the most direct experimental approach to the dynamic properties of the DNA helix. Hydrogen exchange has frequently been used to study the conformations of proteins. The results of such measurements have been interpreted on the basis that a potentially exchangeable hydrogen atom which is involved in intramolecular hydrogen bonding (and/or buried in the interior of a macromolecule) exchanges much more slowly with solvent hydrogen than one not so constrained. With most proteins conditions of exchange can be found for which rates of exchange are measured in hours, or even days. By analogy, the potential applicability of hydrogen exchange methods to a study of hydrogen-bonding and conformation in DNA has been recognized for some time. However, Bradbury et alt. found that essentially complete hydrogen exchange occurred during the 3 min required for sample manipulation following the transfer of a fully deuterated DNA film from a D20 to an H20 environment. As a result of this experience and others (unpublished), it has been generally accepted that the hydrogens of DNA exchange instantaneously and that the methods usually applied to proteins cannot be used. Recently, Englander' has developed a new experimental approach to hydrogen exchange. In this method the macromolecule of interest is incubated to equilibrium with tritiated water, and then quickly separated from unbound tritiumn by gel filtration. The bound tritium is then measured by liquid scintillation counting. This technique has several advantages over the nmore conventional hydrogen exchange methods: it is a true tracer method which does not involve the massive substitution with deuterium characteristic of the older methods, no drying steps are involved so that the macromolecule is kept under controlled environmental conditions at all times, and the method is quite fast, complete separation of the labeled macromolecule from free background tritium being attained in a little over I min. In this paper we demonstrate that under appropriate conditions the exchange of structural hydrogens in DNA (i.e., those involved in interchain hydrogen bonding) can be measured with considerable precision using the tritium-gel filtration tech363