Pommier Y, Mattern M R, Schwartz R E, Zwelling L A
Biochemistry. 1984 Jun 19;23(13):2922-7. doi: 10.1021/bi00308a011.
The sedimentation of DNA-nuclear protein complexes in 1.9 M salt-neutral sucrose gradients (nucleoid sedimentation) was used to examine the effects of the DNA intercalator 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) on mouse leukemia cell DNA. Mild detergent cell lysis and neutral pH make nucleoid sedimentation an extremely gentle, but sensitive, method to detect DNA scission. DNA breaks reduce the compaction of nucleoids and slow their sedimentation. Nucleoids from m-AMSA-treated cells sedimented as did those from untreated cells, indicating no detectable m-AMSA-dependent alterations in compaction despite an apparent underlying DNA break frequency of approximately 3 per 10(6) nucleotides, as measured by alkaline elution with proteinase. Mild proteinase digestion of cell lysates prior to nucleoid sedimentation unmasked some, but not all, of the underlying breaks. The frequency of DNA-protein cross-links in nucleoids from cells treated with m-AMSA was comparable to the single-strand break frequency produced by m-AMSA in whole cells. These results indicate that m-AMSA-induced DNA-protein cross-links conceal DNA breaks so as to prevent swiveling around the breaks within the nucleoids. This unique sort of DNA scission is consistent with the involvement of topoisomerases in the DNA breaks elicited by intercalators in mammalian cells.
利用DNA-核蛋白复合物在1.9M盐中性蔗糖梯度中的沉降作用（核仁沉降）来检测DNA嵌入剂4'-（9-吖啶基氨基）甲磺基间氨基苯甲醚（m-AMSA）对小鼠白血病细胞DNA的影响。温和的去污剂细胞裂解和中性pH值使核仁沉降成为一种极其温和但灵敏的检测DNA断裂的方法。DNA断裂会降低核仁的紧密程度并减缓其沉降速度。m-AMSA处理细胞的核仁沉降情况与未处理细胞的核仁沉降情况相同，这表明尽管用蛋白酶进行碱性洗脱测得每10(6)个核苷酸中约有3个潜在的DNA断裂频率，但未检测到m-AMSA依赖的紧密程度改变。在核仁沉降之前对细胞裂解物进行温和的蛋白酶消化揭示了一些但不是所有潜在的断裂。用m-AMSA处理的细胞的核仁中DNA-蛋白质交联的频率与m-AMSA在全细胞中产生的单链断裂频率相当。这些结果表明，m-AMSA诱导的DNA-蛋白质交联掩盖了DNA断裂，从而防止核仁内围绕断裂处的旋转。这种独特的DNA断裂类型与拓扑异构酶参与哺乳动物细胞中嵌入剂引发的DNA断裂是一致的。
