Structure of the replicative form of bacteriophage φX174: VI. Studies on alkali-denatured double-stranded φX DNA

Abstract

Double-stranded φX DNA which accumulates after infection with bacteriophage φX174 in the presence of chloramphenicol consists mainly of twisted circular double-stranded DNA with no single-strand breaks (component I) and of circular double-stranded DNA, in which single-strand breaks are present (component II). Component II in m-NaCl is denatured with alkali within a sharp pH range, but component I is denatured over a wide pH range (pH 11.2 to 12.4). The midpoints of the transition in m-NaCl are pH 11.3 and 11.9, respectively. Component I in 0.1 m-phosphate is irreversibly denatured at pH 12.7 to 12.8 or higher, giving rise to a structure which sediments in neutral m-NaCl with an S 20,w = 40 s. The configuration of denatured double-stranded φX DNA has been studied by means of velocity sedimentation, isopycnic centrifugation in CsCl and spectrophotometry, and also the reactivity for formaldehyde has been determined. From the results it is concluded that denatured double-stranded φX DNA occurs as a highly twisted structure in which all or almost all hydrogen bonds have been disrupted. Denatured double-stranded φX DNA does not renature at high temperatures in low or high salt concentrations, but is converted spontaneously to native double-stranded DNA after introduction of a single-strand break by pancreatic DNase. The implications of these findings for the structure of denatured double-stranded DNA are discussed.