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

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 degrees C, or after 2--3 h at 37 degrees 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 degrees and 37 degrees C, respectively. The problem of obtaining quantitative data on phosphotriester concentrations from results of alkaline sucrose gradient experiments has been discussed.