Mutagenesis of 8-oxoguanine adjacent to an abasic site in simian kidney cells:: Tandem mutations and enhancement of G→T transversions
Mutagenesis of 8-oxoguanine adjacent to an abasic site in simian kidney cells:: Tandem mutations and enhancement of G→T transversions
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DOI：
10.1021/tx050119r
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发表时间：
2005-08-01
影响因子：
4.1
通讯作者：
Basu, AK
中科院分区：
文献类型：
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作者：
Kalam, MA;Basu, AK
簇状DNA损伤是电离辐射的公认特征。作为同一DNA链上的模型聚集性损伤，我们利用噬菌体载体评估了8-氧鸟嘌呤(8-oxoG)与尿嘧啶相邻的猴肾细胞的诱变潜力。尿嘧啶残基将在体内被尿嘧啶DNA糖基酶切割，产生一个碱性位点(AP位点)。在GUGTC或GTGUC序列上下文中的一个单独的尿嘧啶提供了含有GTGTC的60%的后代，表明发生了与AP位点相反的DAMP掺入或尿嘧啶，但>；30%的群体显示U被A、C或G取代，这表明在A-P位点对面也分别发生了DTMP、dGMP或dCMP掺入。虽然GUG位点对靶向碱基替换的偏好是T>；>；C>；A>；G，但在GUC位点也是如此。我们得出结论，与A-P位点相反的碱基掺入是序列依赖的。对于8-oxoG，与在TG(8-oxo)T序列背景下来自单个8-oxoG的23-24%G-gt；T突变体相比，串联病变UG(8-oxo)T和TG(8-oxo)U分别产生类似于不包含TGT序列的60%和85%的后代。当尿嘧啶与8-oxoG相邻时，检测到相当一部分串联突变。我们发现最有趣的是，包括TG(8-oxo)U位点的单个和串联突变在内的总的靶向G(8-oxo->；)T颠换比例接近60%，而在UG(8-oxo)T位点的比例约为30%。TG(8-oxo)U序列上较高的突变频率可能是因为DNA聚合酶的变化更容易出错。热熔融实验表明，12-聚体中TG(8-oxo)(AP*)序列中8-oxoG：C对的T-m低于Delta G为0.8kcal/mol的(AP*)G(8-oxo)T12-mer中的8-oxoG：C对(其中AP*代表四氢呋喃，模型碱性中心)。当将每个序列中的8-oxoG：C对与8-oxoG：A对进行比较时，发现前者比后者更稳定。在TG(8-oxo)(AP*)12-聚双链中，Delta G为1.6kcal/mol的(AP*)G(8-oxo)T12-mer双链中，C优先于A而不是8-oxoG。这表明，在TG(8-oxo)(AP*)序列中，相对于(AP*)G(8-oxo)T，聚合酶对DAMP的识别效率较低。此外，在TG(8-oxo)(AP*)序列中，错配修复蛋白识别和切除相反的8-oxoG的效率可能会受到影响。
Clustered DNA damages are well-established characteristics of ionizing radiation. As a model clustered lesion in the same strand of DNA, we have evaluated the mutagenic potential of 8-oxoguanine (8-oxoG) adjacent to a uracil in simian kidney cells using a phagemid vector. The uracil residue would be excised by the enzyme uracil DNA glycosylase in vivo generating an abasic site (AP site). A solitary uracil in either GUGTC or GTGUC sequence context provided > 60% progeny containing GTGTC indicating that dAMP incorporation opposite the AP site or uracil occurred, but a > 30% population showed replacement of U by A, C, or G, which suggests that dTMP, dGMP, or dCMP incorporation also occurred, respectively, opposite the A-P site. While the preference for targeted base substitutions at the GUG site was T >> C > A > G, the same at the GUC site was T >> A > C > G. We conclude that base incorporation opposite an A-P site is sequence-dependent. For 8-oxoG, as compared to 23-24% G -> T mutants from a single 8-oxoG in a TG(8-oxo)T sequence context, the tandem lesions UG(8-oxo)T and TG(8-oxo)U generated similar to 60 and > 85% progeny, respectively, that did not contain the TGT sequence. A significant fraction of tandem mutations were detected when uracil was adjacent to 8-oxoG. What we found most interesting is that the total targeted G(8-oxo ->)T transversions that included both single and tandem mutations at the TG(8-oxo)U site was nearly 60% relative to about 30% at the UG(8-oxo)T site. A higher mutational frequency at the TG(8-oxo)U sequence may arise from a change in DNA polymerase that is more error prone. Thermal melting experiments showed that the T-m for the 8-oxoG:C pair in the TG(8-oxo)(AP*) sequence in a 12-mer was lower than the same in a (AP*)G(8-oxo)T 12-mer with Delta Delta G 0.8 kcal/mol (where AP* represents tetrahydrofuran, the model abasic site). When the 8-oxoG:C pair in each sequence was compared with a 8-oxoG:A pair, the former was found to be more stable than the latter. The preference for C over A opposite 8-oxoG for the (AP*)G(8-oxo)T 12-mer duplex with a Delta Delta G of 1.6 kcal/mol dropped to 0.4 kcal/mol in the TG(8-oxo)(AP*) 12-mer duplex. This suggests that the polymerase discrimination to incorporate dCMP over dAMP would be less efficient in the TG(8-oxo)(AP*) sequence relative to (AP*)G(8-oxo)T. Additionally, the efficiency of recognition and excision of A opposite 8-oxoG by a mismatch repair protein may be impaired in the TG(8-oxo)(AP*) sequence context.