Resistance-Guided Mining of Bacterial Genotoxins Defines a Family of DNA Glycosylases

Unique DNA repair enzymes that provide self-resistance against therapeutically important, genotoxic natural products have been discovered in bacterial biosynthetic gene clusters (BGCs). Among these, the DNA glycosylase AlkZ is essential for azinomycin B production and belongs to the HTH_42 superfamily of uncharacterized proteins. Despite their widespread existence in antibiotic producers and pathogens, the roles of these proteins in production of other natural products are unknown. Here, we determine the evolutionary relationship and genomic distribution of all HTH_42 proteins from Streptomyces and use a resistance-based genome mining approach to identify homologs associated with known and uncharacterized BGCs. We find that AlkZ-like (AZL) proteins constitute one distinct HTH_42 subfamily and are highly enriched in BGCs and variable in sequence, suggesting each has evolved to protect against a specific secondary metabolite. As a validation of the approach, we show that the AZL protein, HedH4, associated with biosynthesis of the alkylating agent hedamycin, excises hedamycin-DNA adducts with exquisite specificity and provides resistance to the natural product in cells. We also identify a second, phylogenetically and functionally distinct subfamily whose proteins are never associated with BGCs, are highly conserved with respect to sequence and genomic neighborhood, and repair DNA lesions not associated with a particular natural product. This work delineates two related families of DNA repair enzymes—one specific for complex alkyl-DNA lesions and involved in self-resistance to antimicrobials and the other likely involved in protection against an array of genotoxins—and provides a framework for targeted discovery of new genotoxic compounds with therapeutic potential.