Department of Biology, Portland State University, Portland, Oregon, USA.
J Bacteriol. 2023 Jun 27;205(6):e0012623. doi: 10.1128/jb.00126-23. Epub 2023 May 30.
DNA interstrand cross-links, such as those formed by psoralen-UVA irradiation, are highly toxic lesions in both humans and bacteria, with a single lesion being lethal in Escherichia coli. Despite the lack of effective repair, human cancers and bacteria can develop resistance to cross-linking treatments, although the mechanisms of resistance remain poorly defined. Here, we subjected E. coli to repeated psoralen-UVA exposure to isolate three independently derived strains that were >10,000-fold more resistant to this treatment than the parental strain. Analysis of these strains identified gain-of-function mutations in the transcriptional regulator AcrR and the alpha subunit of RNA polymerase that together could account for the resistance of these strains. Resistance conferred by the AcrR mutation is mediated at least in part through the regulation of the AcrAB-TolC efflux pump. Resistance via mutations in the alpha subunit of RNA polymerase occurs through a still-uncharacterized mechanism that has an additive effect with mutations in AcrR. Both and mutations reduced cross-link formation . We discuss potential mechanisms in relation to the ability to repair and survive interstrand DNA cross-links. Psoralen DNA interstrand cross-links are highly toxic lesions with antimicrobial and anticancer properties. Despite the lack of effective mechanisms for repair, cells can become resistant to cross-linking agents through mechanisms that remain poorly defined. We derived resistant mutants and identified that two gain-of-function mutations in AcrR and the alpha subunit of RNA polymerase confer high levels of resistance to E. coli treated with psoralen-UVA. Resistance conferred by AcrR mutations occurs through regulation of the AcrAB-TolC efflux pump, has an additive effect with RNA polymerase mutations, acts by reducing the formation of cross-links , and reveals a novel mechanism by which these environmentally and clinically important agents are processed by the cell.
DNA 链间交联物，如补骨脂素-UVA 照射形成的交联物，在人和细菌中都是高度毒性的损伤物，单个损伤物在大肠杆菌中是致命的。尽管缺乏有效的修复，人类癌症和细菌可以对交联处理产生抗性，尽管抗性机制仍未得到很好的定义。在这里，我们让大肠杆菌反复接受补骨脂素-UVA 照射，以分离出三种独立衍生的菌株，它们对这种处理的抗性比亲本菌株高出 10000 倍以上。对这些菌株的分析确定了转录调节剂 AcrR 和 RNA 聚合酶的α亚基的功能获得性突变，这些突变共同解释了这些菌株的抗性。AcrR 突变赋予的抗性至少部分是通过调节 AcrAB-TolC 外排泵来介导的。RNA 聚合酶α亚基的突变导致的抗性通过一种尚未确定的机制发生，该机制与 AcrR 突变具有附加效应。 和 突变都减少了交联的形成 。我们讨论了与修复和存活链间 DNA 交联相关的潜在机制。 补骨脂素 DNA 链间交联物是具有抗菌和抗癌特性的高度毒性损伤物。尽管缺乏有效的修复机制，但细胞可以通过仍未明确的机制对交联剂产生抗性。我们衍生出抗性突变体，并确定 AcrR 和 RNA 聚合酶α亚基的两个功能获得性突变赋予大肠杆菌经补骨脂素-UVA 处理后高水平的抗性。AcrR 突变赋予的抗性是通过调节 AcrAB-TolC 外排泵来实现的，与 RNA 聚合酶突变具有相加效应，通过减少交联的形成 来发挥作用，并揭示了这些在环境和临床上重要的药物被细胞处理的新机制。
