Limoli C L, Ward J F
Department of Radiology, University of California, San Diego, La Jolla 92093.
Radiat Res. 1993 May;134(2):160-9.
A novel method is used to introduce double-strand breaks into cellular DNA containing controlled levels of 5-bromo-2'-deoxyuridine (BrdU). Chinese hamster V79 cells substituted with BrdU are treated with Hoechst dye #33258 and then exposed to UVA light. Using neutral elution (pH 7.2) the yield of DNA double-strand breaks is found to be linearly dependent on the level of BrdU substitution (0.36-7.5%), concentration of Hoechst dye (0-100 micrograms cm-3), and fluence of UVA light (0.2-8 kJ m-2). The yield of double-strand breaks produced by this photolysis treatment is 5.1 x 10(-6) breaks/BrdU residue/kJ m-2, regardless of whether one or both strands of the DNA polymer contain BrdU. No double-strand breaks are detected in the absence of Hoechst dye, BrdU, or UVA light. The formation of double-strand breaks appears to involve strand cleavage at a BrdU site on one strand with cleavage in the opposite strand not necessarily requiring the presence of BrdU. The utility of this photolytic regimen in modeling the biological significance of double-strand break lesions and some putative mechanisms for their formation are discussed.
一种新方法被用于将双链断裂引入含有可控水平5-溴-2'-脱氧尿苷（BrdU）的细胞DNA中。用BrdU替代的中国仓鼠V79细胞先用Hoechst染料#33258处理，然后暴露于UVA光下。使用中性洗脱（pH 7.2）发现DNA双链断裂的产率与BrdU替代水平（0.36 - 7.5%）、Hoechst染料浓度（0 - 100微克/立方厘米）和UVA光通量（0.2 - 8千焦/平方米）呈线性相关。无论DNA聚合物的一条链还是两条链都含有BrdU，这种光解处理产生的双链断裂产率为5.1×10⁻⁶个断裂/BrdU残基/千焦/平方米。在没有Hoechst染料、BrdU或UVA光的情况下未检测到双链断裂。双链断裂的形成似乎涉及一条链上BrdU位点的链切割，而另一条链上的切割不一定需要BrdU的存在。讨论了这种光解方案在模拟双链断裂损伤的生物学意义及其形成的一些推测机制方面的实用性。
