The rate of hydrolysis of methyl phosphotriesters in DNA under conditions used in alkaline sucrose gradients

Abstract

Methyl phosphotriesters have been introduced into DNA, in vitro, by reaction with N-methyl- N-nitrosourea and the rate of degradation in alkali has been followed by measurements of the mean sedimentation coefficient using an analytical ultracentrifuge. In 0.3 M NaOH 0.7 M NaCl , a solution commonly used in alkaline sucrose gradient experiments, hydrolysis of the methyl phosphotriesters present is complete after 15 h at 20°C, or after 2–3 h at 37°C. In addition to breaks formed by the latter reaction there was a continuous background degradation of the DNA giving rise to 6.3 and 63 breaks 10 6 nucleotides per h at 20° and 37°C, respectively. The problem of obtaining quantitative data on phosphotriester concentrations from results of alkaline sucrose gradient experiments has been discussed.