Analysis of the effect of DNA alkylation on alkaline elution.
Analysis of the effect of DNA alkylation on alkaline elution.
复制标题
DNA烷基化对碱性洗脱的影响分析。
作者：
A. Fornace;P. P. Dobson;T. Kinsella
最近，有报道称，甲基甲烷磺酸(MMS)处理的细胞中DNA高阶结构和DNA相关蛋白结构的变化可以影响碱洗脱速率，而不依赖于DNA单链断裂(SSB)的频率。这一结论是基于观察到，当细胞裂解物在碱性洗脱之前在pH为10的较长时间内保持时，烷基化DNA的洗脱率增加；这种洗脱率的增加归因于核蛋白结构的变化，而不是额外的DNA SSB。我们更详细地研究了这一现象，发现有证据表明，在pH为10的烷基化DNA中，DNA SSB和碱性不稳定部位都是自发产生的。MMS处理的细胞在pH 10孵育48小时后，DNA被碱性洗脱，pH 12.8的洗脱速度明显高于pH 12.2的洗脱速度。由于在对照细胞DNA中没有观察到这种pH效应，它被归因于在pH为10的孵育过程中产生了碱性不稳定的部位。用碱性解旋法测定DNA单链断裂时，在pH 10孵育48h的烷基化DNA中检测到比0.25h多的SSB，在体外用MMS处理双链DNA并在pH 10孵育48h后进行中性或碱性凝胶电泳分析，在pH 10孵育的双链DNA中检测到明显更高水平的DNA SSB。为了确定DNA结构的变化是否会影响洗脱速度，而不依赖于SSB频率，我们尝试设计了一个实验，该实验将严重破坏DNA的高阶结构，但不会在两条互补的DNA链中的一条中产生DNA SSB。V79细胞在溴脱氧尿嘧啶(BUDR)中生长，所有未被取代的链都与BUDR取代的DNA碱基配对。用碱洗脱法分析DNA时，单线取代的BUDR链在碱中水解并从过滤器中洗脱，这对未取代链的洗脱率没有影响。这些结果表明，碱洗脱率增加的唯一已知原因是DNA SSB；DNA高阶结构的严重变化，包括用碱不稳定的碱基类似物单线取代使单链水解，对未取代链的洗脱率没有影响。
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.