Ljungman M, Hanawalt P C
Department of Biological Sciences, Stanford University, California 94305-5020.
Mol Carcinog. 1992;5(4):264-9. doi: 10.1002/mc.2940050406.
The role of histones and higher order chromatin structures in protecting against oxidative DNA damage was investigated using an in vitro system consisting of nuclear and nucleoid monolayers as model chromatin substrates. These substrates, derived from human skin fibroblasts, were challenged with hydroxyl radicals produced via a Fenton reaction involving Fe(II)-ethylenediaminetetraacetic acid and ascorbic acid. The resulting DNA strand breaks were measured using the alkaline unwinding technique. The sequential removal of chromosomal proteins from the DNA by pretreating nuclear monolayers with increasing concentrations of salt dramatically increased the frequency of hydroxyl radical-induced DNA strand breaks. Furthermore, the DNA in decondensed chromatin was found to contain 14-fold fewer DNA strand breaks than naked, supercoiled DNA, whereas the DNA of "native" chromatin and "condensed" chromatin contained 100-fold and 300-fold fewer breaks, respectively. We conclude that the binding of histones to the DNA and its organization into higher order chromatin structures dramatically protects the DNA against hydroxyl radical-induced DNA strand breaks and thus should be considered part of the cellular defense against the induction of oxidative DNA damage.
利用由细胞核和类核单层组成的体外系统作为模型染色质底物，研究了组蛋白和高阶染色质结构在防止氧化性DNA损伤中的作用。这些源自人皮肤成纤维细胞的底物，受到由涉及Fe(II)-乙二胺四乙酸和抗坏血酸的芬顿反应产生的羟基自由基的攻击。使用碱性解旋技术测量由此产生的DNA链断裂。通过用浓度不断增加的盐预处理细胞核单层，从DNA上依次去除染色体蛋白，显著增加了羟基自由基诱导的DNA链断裂频率。此外，发现解聚染色质中的DNA所含的DNA链断裂比裸露的超螺旋DNA少14倍，而“天然”染色质和“凝聚”染色质的DNA所含的断裂分别少100倍和300倍。我们得出结论，组蛋白与DNA的结合及其组织成高阶染色质结构可显著保护DNA免受羟基自由基诱导的DNA链断裂，因此应被视为细胞防御氧化性DNA损伤诱导的一部分。
