Characterization of the Mutagenic Spectrum of 4-Nitroquinoline 1-Oxide (4-NQO) inAspergillus nidulansby Whole Genome Sequencing

Abstract

4-Nitroquinoline 1-oxide (4-NQO) is a highly carcinogenic chemical that induces mutations in bacteria, fungi, and animals through the formation of bulky purine adducts. 4-NQO has been used as a mutagen for genetic screens and in both the study of DNA damage and DNA repair. In the model eukaryote Aspergillus nidulans, 4-NQO−based genetic screens have been used to study diverse processes, including gene regulation, mitosis, metabolism, organelle transport, and septation. Early work during the 1970s using bacterial and yeast mutation tester strains concluded that 4-NQO was a guanine-specific mutagen. However, these strains were limited in their ability to determine full mutagenic potential, as they could not identify mutations at multiple sites, unlinked suppressor mutations, or G:C to C:G transversions. We have now used a whole genome resequencing approach with mutant strains generated from two independent genetic screens to determine the full mutagenic spectrum of 4-NQO in A. nidulans. Analysis of 3994 mutations from 38 mutant strains reveals that 4-NQO induces substitutions in both guanine and adenine residues, although with a 19-fold preference for guanine. We found no association between mutation load and mutagen dose and observed no sequence bias in the residues flanking the mutated purine base. The mutations were distributed randomly throughout most of the genome. Our data provide new evidence that 4-NQO can potentially target all base pairs. Furthermore, we predict that current practices for 4-NQO−induced mutagenesis are sufficient to reach gene saturation for genetic screens with feasible identification of causative mutations via whole genome resequencing.

Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus

“…Mutagenesis screens utilizing 4-nitroquinoline 1-oxide (4-NQO) typically produce single nucleotide mutations, with a preference for guanine-to-adenine (G-A) transitions. Estimations of the number of mutations induced by treatment with 4-NQO are dependent on the length of exposure and the subsequent kill rate; however, it has been predicted that current practices using 4-NQO are sufficient to reach saturation of screens (36). One major challenge in using whole-genome sequencing data to find a mutation causing the phenotype of interest is the presence of variants that do not influence that phenotype (background).…”

Discovery of McrA, a master regulator of Aspergillus secondary metabolism

“…Riboflavin prototrophs can be easily selected on media lacking riboflavin. 4-nitroquinoline 1-oxide (NQO), which specifically causes base-pair substitutions (Bailleul et al, 1989;Downes et al, 2014). We used a range of NQO concentrations (0-3.5 lg ml 21 ) and selected mutants in which expression of AfriboB was activated on a medium lacking riboflavin (e.g.…”

No difference in 4‐nitroquinoline induced tumorigenesis between germ‐free and colonized mice

“…4NQO is metabolically converted to 4-hydroxyaminoquinolone 1-oxide, which induces gene mutations through the formation of bulky adducts on purines. 27,28 Since we found no difference in the number and size of tumors in germ-free and conventionally housed mice, our data suggests that the oral microbiome does not play a role in altering the mutagenic properties of 4NQO. Our study followed a standard protocol for 4NQO induced oral cancer with a follow-up time of 10 weeks after the end of treatment.…”

“…There was no statistically significant difference in the number and size of tumors between germ‐free and conventionally housed mice ( P > 0.05). 4NQO is metabolically converted to 4‐hydroxyaminoquinolone 1‐oxide, which induces gene mutations through the formation of bulky adducts on purines . Since we found no difference in the number and size of tumors in germ‐free and conventionally housed mice, our data suggests that the oral microbiome does not play a role in altering the mutagenic properties of 4NQO.…”