Residual activity of denatured transforming DNA of Haemophilus influenzae: A naturally occurring cross-linked DNA

Abstract

The residual activity of denatured transforming DNA of Haemophilus influenzae is accounted for by the presence of cross-linked molecules in all DNA preparations, whereas single-stranded DNA has no detectible transforming activity. The specific transforming activity of the cross-linked molecules is at least 90% that of native DNA. This conclusion is reached from the observation that the residual activity sediments 1.3 to 1.4 times faster than the bulk of the denatured DNA through an alkaline sucrose gradient, almost identical to the rate of an artificially cross-linked DNA. Similarly, the residual activity is eluted from a Sepharose column 1.5 times faster than the bulk of the denatured DNA and almost identical to the elution pattern of added native DNA of the same homogeneous size. Moreover, the buoyant density in CsCl of the molecules carrying the residual activity was found to be close to that of native DNA and considerably lighter than that of denatured DNA; with denatured biological hybrid ( 15 N 2 H 14 N 1 H ) DNA, this fraction was found only at a buoyant density slightly heavier than native hybrid DNA; no activity was found near the densities of native or denatured heavy DNA or native light DNA. The intrinsic residual activity is equal for all melting conditions tried and for nine different markers studied. It was found to be 11.5% for a DNA preparation with an average molecular weight of 30 × 10 6 daltons, and it is linearly related to the molecular weight of the DNA. Therefore, it is estimated that about three cross-links are present per bacterial genome if they pre-exist in vivo.