Effect of episomally encoded DNA polymerases on chemically induced mutagenesis at the hisG46 target in Ames test
Effect of episomally encoded DNA polymerases on chemically induced mutagenesis at the hisG46 target in Ames test
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DOI：
10.1186/s41021-020-00154-2
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发表时间：
2020-03-24
影响因子：
1.7
通讯作者：
Honma, Masamitsu
中科院分区：
文献类型：
--
作者：
Gruz, Petr;Shimizu, Masatomi;Honma, Masamitsu
背景标准艾姆斯试验菌株对化学和物理诱变剂的高敏感性归因于由mucAB操纵子编码的附加型Y家族DNA聚合酶RI。色葡萄鼠伤寒试验菌株还在60-kDa隐蔽质粒上携带另一个相关的samAB操纵子。与染色体编码的Y家族DNA聚合酶V和IV相反，这些质粒产生的聚合酶基因在哺乳动物细胞中没有直接对应物。通过复制受损的模板，DNA聚合酶在诱变和基因组稳定性中发挥核心作用。因此，有必要研究它们的特异性，以了解原核与真核（哺乳动物）系统之间的诱变差异。为此，我们分离并分别表达了由mucAB和samAB操纵子编码的DNA聚合酶亚基。在展示了这些酶如何控制标准hisD 3052和hisG 428艾姆斯测试靶标的化学和紫外线突变后，我们现在将第三个艾姆斯测试靶标hisG 46添加到三部曲中。结果构建了4株以hisG 46为靶点的新型艾姆斯试验菌株，它们在lac启动子的控制下，分别表达活化的DNA聚合酶MucA'和SamA'辅助亚基，以及MucB和SamB催化亚基。用这些聚合酶组件取代标准TA 100菌株中存在的内源PolRI、PolV和SamAB聚合酶，并测试它们促进化学诱导诱变的能力。SamA' + SamB能够比SamA' + MucB更高程度地促进AF-2和1，8-DNP诱导的突变。MucA' + MucB（PolRI*）比其野生型对应物更有效地促进MMS以及自发诱变，但对于包括AFB 1的其它诱变剂效率较低。PolRI和SamA ′ B显著抑制叠氮化物诱变。结论建立了一种新的非SOS依赖性的hisG 46艾姆斯试验背景下DNA聚合酶RI和SamA 'B及其嵌合体的过表达系统，并用几种典型的诱变剂进行了验证。总体而言，TA 100菌株对大多数受试诱变剂的敏感性最高。所观察到的PolRI* 对叠氮化物诱变的抑制表明，这种类型的Y家族DNA聚合酶也可以对受损的DNA进行“校正性”无错误复制。
Background The standard Ames test strains owe their high sensitivity to chemical and physical mutagens to the episomal Y-family DNA polymerase RI encoded by the mucAB operon. The S. typhimurium test strains carry also another related samAB operon on a 60-kDa cryptic plasmid. In contrast to the chromosomally encoded Y-family DNA polymerases V and IV, these plasmid born polymerase genes have no direct counterpart in mammalian cells. By replicating damaged templates, DNA polymerases play a central role in mutagenesis and genome stability. It is therefore imperative to investigate their specificity to understand differences in mutagenesis between the prokaryotic versus eukaryotic (mammalian) systems. To this end we have isolated and separately expressed the DNA polymerase subunits encoded by the mucAB and samAB operons. After demonstrating how these enzymes control chemical and UV mutagenesis at the standard hisD3052 and hisG428 Ames test targets, we are now adding the third Ames test target hisG46 to the trilogy. Results Four new Ames tester strains based on the hisG46 target have been constructed expressing the activated DNA polymerase MucA' and SamA' accessory subunits combined with the MucB and SamB catalytical subunits under the control of lac promoter. These polymerase assemblies were substituted for the endogenous PolRI, PolV and SamAB polymerases present in the standard TA100 strain and tested for their abilities to promote chemically induced mutagenesis. SamA' + SamB has been able to promote mutagenesis induced by AF-2 and 1,8-DNP to higher extent than SamA' + MucB. The MucA' + MucB (PolRI*) more efficiently promoted MMS as well as spontaneous mutagenesis than its wild type counterpart but was less efficient for other mutagens including AFB1. Strikingly azide mutagenesis was inhibited by PolRI and also SamA'B. Conclusion A new system for SOS-independent overexpression of the activated DNA polymerases RI and SamA'B and their chimeras in the hisG46 Ames test background has been established and validated with several representative mutagens. Overall, the TA100 strain showed the highest sensitivity towards most tested mutagens. The observed inhibition of azide mutagenesis by PolRI* suggests that this type of Y-family DNA polymerases can perform also "corrective" error free replication on a damaged DNA.