On the chemical yield of base lesions, strand breaks, and clustered damage generated in plasmid DNA by the direct effect of X rays
On the chemical yield of base lesions, strand breaks, and clustered damage generated in plasmid DNA by the direct effect of X rays
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DOI：
10.1667/rr0964.1
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发表时间：
2007-09-01
影响因子：
3.4
通讯作者：
Bernhard, William A.
中科院分区：
文献类型：
--
作者：
Purkayastha, Shubhadeep;Milligan, Jamie R.;Bernhard, William A.
本研究的目的是确定由于电离辐射的直接影响而导致的DNA碱基损伤、脱氧核糖损伤和聚集性损伤的产量，并将这些与先前在同一pUC18质粒中测量的DNA捕获自由基的产量进行比较。质粒制备成水合范围为2.5 < r < 22.5 mol水/mol核苷酸的膜。琼脂糖凝胶电泳检测单链断裂（SSBs）和双链断裂（DSBs）。利用碱基切除修复酶内切酶III （Nth）和甲胺-多嘧啶- dna糖基化酶（Fpg）将特定类型的碱基病变转化为SSBs和DSBs。与DNA捕获自由基的产率相比，这种方法检测到的碱基损伤产率对水合水平(r)的依赖性明显不同；r在2.5 ~ 22.5范围内，formei下降了3.2倍，r在22.5范围内增加了2.4倍。为了解释这种差异，我们提出，通过直接效应在质粒DNA中产生的SSB产量不能用泊松过程进行适当分析，泊松过程假设每个质粒平均有一条链断裂，并忽略了在质粒中单个轨道产生多个SSB的可能性。另一方面，dsb的产率与水化作用下自由基捕获的变化是一致的。因此，这些集群的组成可以量化。对于完全水合的pUC18，两条相反链上的每一条脱氧核糖损伤的产率为3.5 +/- 0.5 nmol/J，而对于至少一个位点是受损碱基的相反损伤的dsb，产率为4.1 +/- 0.9 nmol/J。(c) 2007年，辐射研究学会。
The purpose of this study was to determine the yield of DNA base damages, deoxyribose damage, and clustered lesions due to the direct effects of ionizing radiation and to compare these with the yield of DNA trapped radicals measured previously in the same pUC18 plasmid. The plasmids were prepared as films hydrated in the range 2.5 < r < 22.5 mol water/mol nucleotide. Single-strand breaks (SSBs) and double-strand breaks (DSBs) were detected by agarose gel electrophoresis. Specific types of base lesions were converted into SSBs and DSBs using the base-excision repair enzymes endonuclease III (Nth) and formami-dopyrimidine-DNA glycosylase (Fpg). The yield of base damage detected by this method displayed a strikingly different dependence on the level of hydration (r) compared with that for the yield of DNA trapped radicals; the formei decreased by 3.2 times as r was varied from 2.5 to 22.5 and the later increased by 2.4 times over the same range. To explain this divergence, we propose that SSB yields produced in plasmid DNA by the direct effect cannot be analyzed properly with a Poisson process that assumes an average of one strand break per plasmid and neglects the possibility of a single track producing multiple SSBs within a plasmid. The yields of DSBs, on the other hand, are consistent with changes in free radical trapping as a function of hydration. Consequently, the composition of these clusters could be quantified. Deoxyribose damage on each of the two opposing strands occurs with a yield of 3.5 +/- 0.5 nmol/J for fully hydrated pUC18, comparable to the yield of 4.1 +/- 0.9 nmol/J for DSBs derived from opposed damages in which at least one of the sites is a damaged base. (c) 2007 by Radiation Research Society.