Muiras M L, Giacomoni P U, Tachon P
Laboratoires de Recherche Fondamentale de L'Oréal, Aulnay-sous-Bois, France.
Mutat Res. 1993 Jan;295(1):47-54. doi: 10.1016/0921-8734(93)90010-z.
The conversion of the covalently closed circular double-stranded supercoiled DNA (pBR322) to a relaxed circle was used to investigate DNA nicking induced by Fe2+ and H2O2. In our experimental conditions of ionic strength (150 mM NaCl), pH = 7 and temperature (37 degrees C), the dose-response curve for the ferrous iron mediated H2O2 dependent DNA nicking is peculiar. For a fixed concentration of ferrous iron (2 microM), the concentration of H2O2 producing a maximum extent of DNA nicking was about 10-30 microM. The DNA single-strand breakage decreased with an increase of H2O2 concentration. We have investigated the effects of several factors such as the nature of the buffer, ionic strength, temperature and pH. Buffer components leading to the autoxidation of ferrous iron to ferric iron (phosphate) or to the scavenging of reactive oxygen species (Tris) greatly alter the dose-response curve. The H2O2 concentrations required for producing the maximum extent of DNA single-strand breaks at 4 degrees C and 56 degrees C were respectively 30 microM and 3 microM. At pH = 10, the pattern of the dose-response curve was totally different. The data showed that the peculiar dose-response curve for the ferrous iron mediated H2O2 dependent DNA nicking greatly depended on the experimental conditions.
利用共价闭合环状双链超螺旋DNA（pBR322）向松弛环的转化来研究Fe2+和H2O2诱导的DNA切口。在我们离子强度为150 mM NaCl、pH = 7和温度为37℃的实验条件下，亚铁介导的H2O2依赖性DNA切口的剂量反应曲线很奇特。对于固定浓度的亚铁（2 microM），产生最大程度DNA切口的H2O2浓度约为10 - 30 microM。DNA单链断裂随着H2O2浓度的增加而减少。我们研究了几种因素的影响，如缓冲液的性质、离子强度、温度和pH。导致亚铁自氧化为铁离子（磷酸盐）或清除活性氧（Tris）的缓冲液成分极大地改变了剂量反应曲线。在4℃和56℃产生最大程度DNA单链断裂所需的H2O2浓度分别为30 microM和3 microM。在pH = 10时，剂量反应曲线的模式完全不同。数据表明，亚铁介导的H2O2依赖性DNA切口的奇特剂量反应曲线很大程度上取决于实验条件。
