New method for estimating clustering of DNA lesions induced by physical/chemical mutagens using fluorescence anisotropy
New method for estimating clustering of DNA lesions induced by physical/chemical mutagens using fluorescence anisotropy
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DOI：
10.1016/j.ab.2017.08.007
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发表时间：
2017-11-01
影响因子：
2.9
通讯作者：
Saito, Takeshi
中科院分区：
文献类型：
--
作者：
Akamatsu, Ken;Shikazono, Naoya;Saito, Takeshi
我们已经开发了一种新的方法来估计本地化的DNA损伤，如脱嘌呤/脱嘧啶位点（AP）的DNA荧光各向异性。该方法旨在表征由DNA损伤剂如电离辐射和遗传毒性化学品产生的成簇DNA损伤。使用具有氨氧基的荧光探针（AlexaFluor 488）来标记AP。我们通过在酸性条件下加热制备具有AP的pUC 19质粒作为受损DNA的模型，随后标记AP。我们发现，所观察到的荧光各向异性（r（obs））降低平均AP密度（λ（AP）：AP每碱基对的数量）增加，由于homo-FRET，和AP随机分布。我们将这种方法应用于三种DNA损伤剂，Co-60 γ射线，甲磺酸甲酯（MMS）和新制癌素（NCS）。我们发现r（obs）-lambda（AP）关系在MMS和NCS之间存在显著差异。在低AP浓度（λ（AP）< 0.001）时，MMS诱导的AP似乎分布不紧密，而NCS诱导的AP明显聚集。相比之下，由60 Co γ射线诱导的AP聚集类似于随机分布，但可能更容易发生。该方法简便，可用于电离辐射和遗传毒性物质的致突变性评价。(C)2017爱思唯尔公司All rights reserved.
We have developed a new method for estimating the localization of DNA damage such as apurinic/apyrimidinic sites (APs) on DNA using fluorescence anisotropy. This method is aimed at characterizing clustered DNA damage produced by DNA-damaging agents such as ionizing radiation and genotoxic chemicals. A fluorescent probe with an aminooxy group (AlexaFluor488) was used to label APs. We prepared a pUC19 plasmid with APs by heating under acidic conditions as a model for damaged DNA, and subsequently labeled the APs. We found that the observed fluorescence anisotropy (r(obs)) decreases as averaged AP density (lambda(AP): number of APs per base pair) increases due to homo-FRET, and that the APs were randomly distributed. We applied this method to three DNA-damaging agents, Co-60 gamma-rays, methyl methanesulfonate (MMS), and neocarzinostatin (NCS). We found that r(obs)-lambda(AP) relationships differed significantly between MMS and NCS. At low AP density (lambda(AP) < 0.001), the APs induced by MMS seemed to not be closely distributed, whereas those induced by NCS were remarkably clustered. In contrast, the AP clustering induced by 60Co gamma-rays was similar to, but potentially more likely to occur than, random distribution. This simple method can be used to estimate mutagenicity of ionizing radiation and genotoxic chemicals. (C) 2017 Elsevier Inc. All rights reserved.