Resistance-guided mining of bacterial genotoxins defines a family of DNA glycosylases

Unique DNA repair enzymes that provide self-resistance against genotoxic natural products have been discovered recently in bacterial biosynthetic gene clusters (BGCs). The DNA glycosylase AlkZ belongs to a superfamily of uncharacterized proteins found in antibiotic producers and pathogens, but despite its importance to azinomycin B resistance, the roles of AlkZ orthologs in production of other natural products are unknown. Here, we analyze the genomic distribution and use a resistance-based genome mining approach to identify Streptomyces AlkZ homologs associated with known and uncharacterized BGCs. We show that the ortholog associated with synthesis of the alkylating agent hedamycin excises hedamycin-DNA adducts and provides resistance to the genotoxin in cells. Our results define AlkZ in self-resistance to specific antimicrobials and implicate a related but distinct homolog, which we name AlkX, in protection against an array of genotoxins. This work provides a framework for targeted discovery of new genotoxic compounds with therapeutic potential.

Identification of a novel structure-specific endonuclease AziN that contributes to the repair of azinomycin B-mediated DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) induced by the highly genotoxic agent azinomycin B (AZB) can cause severe perturbation of DNA structure and even cell death. However, Streptomyces sahachiroi, the strain that produces AZB, seems almost impervious to this danger because of its diverse and distinctive self-protection machineries. Here, we report the identification of a novel endonuclease-like gene aziN that contributes to drug self-protection in S. sahachiroi. AziN expression conferred AZB resistance on native and heterologous host strains. The specific binding reaction between AziN and AZB was also verified in accordance with its homology to drug binding proteins, but no drug sequestering and deactivating effects could be detected. Intriguingly, due to the high affinity with the drug, AziN was discovered to exhibit specific recognition and binding capacity with AZB-mediated ICL structures, further inducing DNA strand breakage. Subsequent in vitro assays demonstrated the structure-specific endonuclease activity of AziN, which cuts both damaged strands at specific sites around AZB-ICLs. Unravelling the nuclease activity of AziN provides a good entrance point to illuminate the complex mechanisms of AZB-ICL repair.

Dimeric and trimeric derivatives of the azinomycin B chromophore show enhanced DNA binding

Compound1d(Kapp= 2.4 × 107M−1), a derivative of the azinomycin B chromophore, strongly associates with DNA by intercalation and major groove binding.