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ABSTRACT 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 acrR and rpoA mutations reduced cross-link formation in vivo . We discuss potential mechanisms in relation to the ability to repair and survive interstrand DNA cross-links. IMPORTANCE 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 in vivo , and reveals a novel mechanism by which these environmentally and clinically important agents are processed by the cell. 

Recombination mediator proteins have come into focus as promising targets for cancer therapy, with synthetic lethal approaches now clinically validated by the efficacy of PARP inhibitors in treating BRCA2 cancers and RECQ inhibitors in treating cancers with microsatellite instabilities. Thus, understanding the cellular role of recombination mediators is critically important, both to improve current therapies and develop new ones that target these pathways. Our mechanistic understanding of BRCA2 and RECQ began in Escherichia coli. Here, we review the cellular roles of RecF and RecQ, often considered functional homologs of these proteins in bacteria. Although these proteins were originally isolated as genes that were required during replication in sexual cell cycles that produce recombinant products, we now know that their function is similarly required during replication in asexual or mitotic-like cell cycles, where recombination is detrimental and generally not observed. Cells mutated in these gene products are unable to protect and process replication forks blocked at DNA damage, resulting in high rates of cell lethality and recombination events that compromise genome integrity during replication. 

Abstract UV irradiation induces pyrimidine dimers that block polymerases and disrupt the replisome. Restoring replication depends on the recF pathway proteins which process and maintain the replication fork DNA to allow the lesion to be repaired before replication resumes. Oxidative DNA lesions, such as those induced by hydrogen peroxide (H2O2), are often thought to require similar processing events, yet far less is known about how cells process oxidative damage during replication. Here we show that replication is not disrupted by H2O2-induced DNA damage in vivo. Following an initial inhibition, replication resumes in the absence of either lesion removal or RecF-processing. Restoring DNA synthesis depends on the presence of manganese in the medium, which we show is required for replication, but not repair to occur. The results demonstrate that replication is enzymatically inactivated, rather than physically disrupted by H2O2-induced DNA damage; indicate that inactivation is likely caused by oxidation of an iron-dependent replication or replication-associated protein that requires manganese to restore activity and synthesis; and address a long standing paradox as to why oxidative glycosylase mutants are defective in repair, yet not hypersensitive to H2O2. The oxygen-sensitive pausing may represent an adaptation that prevents replication from occurring under potentially lethal or mutagenic conditions.