Chiu S M, Xue L Y, Friedman L R, Oleinick N L
Department of Radiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.
Biochemistry. 1995 Feb 28;34(8):2653-61. doi: 10.1021/bi00008a032.
The induction of DNA DSB (double-strand breaks) in isolated nuclear chromatin by Cu(II) or Fe(II)-EDTA in the presence of H2O2 and ascorbate has been compared to DSB induction by gamma-radiation. V79 nuclei embedded in agarose plugs were treated with each agent on ice, and the resultant DNA fragments were analyzed by pulsed-field gel electrophoresis. In the absence of low molecular weight radical scavengers, both irradiation and treatment with iron ion induced random DSB, as judged by the size distribution of DNA fragments, and the yield of DSB in each case was enhanced by either the expansion of chromatin (approximately 5-fold) or the removal of histones (21-25-fold) before treatment. In contrast, treatment with Cu(II) produced small DNA fragments of uniform size (approximately 100-200 kbp), independent of the yield of DSB. In addition, neither the DNA fragment size nor the yield of DSB produced by Cu(II) was affected by the prior removal of histones from chromatin. Deproteinized DNA was degraded randomly by Cu(II) but at a slower rate than observed for chromatin. In the presence of ascorbate, H2O2 was found to be essential for DSB induction by Fe(II)-EDTA but not by Cu(II), possibly because H2O2 can be produced from ascorbate and Cu(II) in the presence of oxygen. Despite the above differences between the production of DSB by the two metal ions, DSB induction in native chromatin by either metal ion was blocked by 0.1 M EDTA or 0.25 M thiourea but was resistant to the hydroxyl radical scavengers 0.25 M DMSO and 0.25 M mannitol.(ABSTRACT TRUNCATED AT 250 WORDS)
已将在过氧化氢和抗坏血酸盐存在的情况下，铜（II）或铁（II）-乙二胺四乙酸（EDTA）在分离的核染色质中诱导DNA双链断裂（DSB）的情况与γ射线诱导DSB的情况进行了比较。将包埋在琼脂糖凝胶块中的V79细胞核在冰上用每种试剂处理，然后通过脉冲场凝胶电泳分析所得的DNA片段。在没有低分子量自由基清除剂的情况下，根据DNA片段的大小分布判断，辐射和铁离子处理均诱导随机DSB，并且在每种情况下，通过在处理前扩展染色质（约5倍）或去除组蛋白（21 - 25倍），DSB的产量都会增加。相比之下，铜（II）处理产生大小均匀的小DNA片段（约100 - 200 kbp），与DSB的产量无关。此外，铜（II）产生的DNA片段大小和DSB产量均不受染色质预先去除组蛋白的影响。脱蛋白的DNA被铜（II）随机降解，但速度比染色质慢。在存在抗坏血酸盐的情况下，发现过氧化氢对于铁（II）-EDTA诱导DSB是必需的，但对铜（II）不是必需的，这可能是因为在有氧存在的情况下，抗坏血酸盐和铜（II）可以产生过氧化氢。尽管两种金属离子产生DSB的情况存在上述差异，但两种金属离子在天然染色质中诱导DSB均被0.1 M乙二胺四乙酸（EDTA）或0.25 M硫脲阻断，但对羟基自由基清除剂0.25 M二甲基亚砜（DMSO）和0.25 M甘露醇具有抗性。（摘要截断于250字）
