Inter-strand crosslinking of DNA by nitrogen mustard

Abstract

The effect of reaction with nitrogen mustard on the denaturability of DNA was studied in order to clarify the nature of the effect that has been attributed to inter-strand crosslinking. Reaction of bacterial DNA with concentrations of nitrogen mustard as low as 5 × 10 −7 M , corresponding to alkylation of approximately 0·005% of the bases, converted an appreciable fraction of the DNA to a form that did not denature after exposure to dilute sodium hydroxide or to formamide. The hydrogen-bonded structure of the DNA bihelix is broken down by such exposures, but the altered fraction of the DNA reverts to the bihelical form after removal of the denaturant. (Unreaeted DNA remains almost completely denatured after these exposures.) Several physical properties of this recovered bihelical DNA were measured and were found to be similar to those of the original DNA. Measurements of the fraction of the DNA rendered resistant to denaturation were performed by CsCl density-gradient equilibrium ultracentrifugation. Studies with density-hybrid DNA showed that the denaturation resistance produced by nitrogen mustard treatment correlates with an inhibition of strand separation. When DNA was subjected to a mild HN2† treatment and then exposed to alkali at various temperatures, the fraction of the DNA denatured was found to be constant over a wide temperature range. This indicates that the denaturation resistance is mediated by covalent bonds. Denaturation-resistant fractions were produced by the reaction of DNA with the bifunctional nitrogen mustard, HN2, and with bifunctional epoxides. Mono-functional analogues of these agents did not produce this effect. The requirement for sequential reactions of two functional groups was associated with a time-lag in the appearance of denaturation-resistant molecules during reaction with HN2. The formation of denaturation-resistant DNA was initially first-order with respect to HN2 concentration. This indicates that a denaturation-resistant DNA molecule can be produced by the reaction of a single molecule of HN2. The results indicate that nitrogen mustard generates covalent crosslinks between the paired strands of bihelical DNA, and that a single such crosslink per DNA molecule prevents denaturation. Studies with 14 C-labeled HN2 showed that only a small fraction, about 4%, of the DNA-bound mustard molecules became effective crosslinks, under the reaction conditions employed.