Structure of the replicative form of bacteriophage φX174: VII. Renaturation of denatured double-stranded φX DNA

Abstract

Denatured double-stranded φX DNA (RF DNA) is formed after exposure of component I (covalently closed circular duplex) to alkali and reneutralization of the alkali. Under conditions which are optimal for the renaturation of linear phage DNA, denatured double-stranded φX DNA cannot be renatured. Renaturation will proceed very rapidly, however, in alkaline solution (pH 12) at 50 °C. Breakage of a phosphodiester bond in denatured double-stranded φX DNA will lead to one of the following products, irrespective of the means by which the break is achieved (enzymic hydrolysis, γ-irradiation or incubation with reducing agents): 1. (a) native double-stranded DNA (component II; nicked circular duplex) is formed in buffers of low ionic strength (0.01 m-phosphate—0.001 m-sodium citrate, pH 7) at 0 to 30 °C; 2. (b) single-stranded DNA is formed in buffers of low ionic strength at temperatures exceeding 30 °C; 3. (c) a heterogeneous product, presumably consisting of partially renatured DNA, is formed in buffers of high ionic strength (0.1 m-Tris or m-NaCl). These results suggest that the nature of the product of denatured double-stranded φX DNA, in which a phosphodiester bond is broken, is primarily determined by environmental conditions such as temperature and ionic strength. Cross-linking of the two strands in component I by means of nitrogen mustard prevents the conversion by alkali into denatured RF DNA. On the other hand, interstrand cross-links in denatured RF DNA impede the molecules from renaturing to form component II after chain breakage. These results lend support to a model for the structure of denatured RF DNA which had been proposed earlier. This means that a shift of the two strands with respect to each other and a high number of crossings over of both strands have a major role in the irreversibility of denaturation of component I.