Analysis of the effect of DNA alkylation on alkaline elution

Abstract

Recently, it has been reported that changes in DNA higher-order structure and DNA-associated protein structure in methylmethane sulfonate (MMS)-treated cells can affect the rate of alkaline elution independent of the DNA single-strand break (SSB) frequency. This conclusion was based on the observation that the rate of elution of alkylated DNA increased when the cell lysates were held at pH 10 for extended periods of time prior to alkaline elution; this increase in elution rate was attributed to changes in nucleoprotein structure rather than additional DNA SSB. We have examined this phenomenon in more detail and find evidence that both DNA SSB and alkaline-labile sites are produced spontaneously in alkylated DNA at pH 10. Alkaline elution of DNA from MMS-treated cells was carried out after incubation of the cell lysates at pH 10 for 48 h; the rate of elution was markedly greater at pH 12.8 compared with pH 12.2 elution. Since this pH effect was not seen in control cell DNA, it was attributed to the production of alkaline-labile sites during the pH 10 incubation. When DNA SSB were measured by alkaline unwinding assay, more SSB were detected in alkylated DNA held at pH 10 for 48 h than for 0.25 h. Double-stranded DNA was treated with MMS in vitro and analyzed by neutral or alkaline gel electrophoresis immediately or after 48 h incubation at pH 10; substantially higher levels of DNA SSB were detected with the pH 10 incubation by either neutral or alkaline gel electrophoresis. In order to determine if changes in DNA structure affect the rate of elution independent of the SSB frequency, we attempted to design an experiment which would severely disrupt DNA higher-order structure but not produce DNA SSB in one of the two complementary DNA strands. V79 cells were grown in bromodeoxyurine (BUdR) under conditions such that all unsubstituted strands were base paired with BUdR-substituted DNA. When this DNA was analyzed by alkaline elution, the unifilar substituted BUdR strand hydrolyzed in alkali and eluted from the filter; this had no effect on the elution rate of the unsubstituted strand. These results indicate that the only known cause for an increase in alkaline elution rate is DNA SSB; severe changes in DNA higher order structure including hydrolysis of one strand by unifilar substitution with an alkaline-labile base analog have no effect on the elution rate of the unsubstituted strand.