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TatD enzymes are evolutionarily conserved deoxyribonucleases associated with DNA repair, apoptosis, development, and parasite virulence. Three TatD paralogs exist in humans, but their nuclease functions are unknown. Here, we describe the nuclease activities of two of the three human TatD paralogs, TATDN1 and TATDN3, which represent two phylogenetically distinct clades based on unique active site motifs. We found that in addition to 3′-5′ exonuclease activity associated with other TatD proteins, both TATDN1 and TATDN3 exhibited apurinic/apyrimidinic (AP) endonuclease activity. The AP endonuclease activity was observed only in double-stranded DNA, whereas exonuclease activity was operative primarily in single-stranded DNA. Both nuclease activities were observed in the presence of Mg2+ or Mn2+, and we identified several divalent metal cofactors that inhibited exonuclease and supported AP endonuclease activity. Biochemical analysis and a crystal structure of TATDN1 bound to 2′-deoxyadenosine 5′-monophosphate in the active site are consistent with two-metal ion catalysis, and we identify several residues that differentiate nuclease activities in the two proteins. In addition, we show that the three Escherichia coli TatD paralogs are also AP endonucleases, indicating that this activity is conserved across evolution. Together, these results indicate that TatD enzymes constitute a family of ancient AP endonucleases.

 

Microbes produce a broad spectrum of antibiotic natural products, including many DNA-damaging genotoxins. Among the most potent of these are DNA alkylating agents in the spirocyclopropylcyclohexadienone (SCPCHD) family, which includes the duocarmycins, CC-1065, gilvusmycin, and yatakemycin. The yatakemycin biosynthesis cluster inStreptomycessp. TP-A0356 contains an AlkD-related DNA glycosylase, YtkR2, that serves as a self-resistance mechanism against yatakemycin toxicity. We previously reported that AlkD, which is not present in an SCPCHD producer, provides only limited resistance against yatakemycin. We now show that YtkR2 and C10R5, a previously uncharacterized homolog found in the CC-1065 biosynthetic gene cluster ofStreptomyces zelensis, confer far greater resistance against their respective SCPCHD natural products. We identify a structural basis for substrate specificity across gene clusters and show a correlation between in vivo resistance and in vitro enzymatic activity indicating that reduced product affinity—not enhanced substrate recognition—is the evolutionary outcome of selective pressure to provide self-resistance against yatakemycin and CC-1065.

 

Two families of DNA glycosylases (YtkR2/AlkD, AlkZ/YcaQ) have been found to remove bulky and crosslinking DNA adducts produced by bacterial natural products. Whether DNA glycosylases eliminate other types of damage formed by structurally diverse antibiotics is unknown. Here, we identify four DNA glycosylases—TxnU2, TxnU4, LldU1 and LldU5—important for biosynthesis of the aromatic polyketide antibiotics trioxacarcin A (TXNA) and LL-D49194 (LLD), and show that the enzymes provide self-resistance to the producing strains by excising the intercalated guanine adducts of TXNA and LLD. These enzymes are highly specific for TXNA/LLD-DNA lesions and have no activity toward other, less stable alkylguanines as previously described for YtkR2/AlkD and AlkZ/YcaQ. Similarly, TXNA-DNA adducts are not excised by other alkylpurine DNA glycosylases. TxnU4 and LldU1 possess unique active site motifs that provide an explanation for their tight substrate specificity. Moreover, we show that abasic (AP) sites generated from TxnU4 excision of intercalated TXNA-DNA adducts are incised by AP endonuclease less efficiently than those formed by 7mG excision. This work characterizes a distinct class of DNA glycosylase acting on intercalated DNA adducts and furthers our understanding of specific DNA repair self-resistance activities within antibiotic producers of structurally diverse, highly functionalized DNA damaging agents.

 

ABSTRACT 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. IMPORTANCE Bacteria are rich sources of secondary metabolites that include DNA-damaging genotoxins with antitumor/antibiotic properties. Although Streptomyces produce a diverse number of therapeutic genotoxins, efforts toward targeted discovery of biosynthetic gene clusters (BGCs) producing DNA-damaging agents is lacking. Moreover, work on toxin-resistance genes has lagged behind our understanding of those involved in natural product synthesis. Here, we identified over 70 uncharacterized BGCs producing potentially novel genotoxins through resistance-based genome mining using the azinomycin B-resistance DNA glycosylase AlkZ. We validate our analysis by characterizing the enzymatic activity and cellular resistance of one AlkZ ortholog in the BGC of hedamycin, a potent DNA alkylating agent. Moreover, we uncover a second, phylogenetically distinct family of proteins related to Escherichia coli YcaQ, a DNA glycosylase capable of unhooking interstrand DNA cross-links, which differs from the AlkZ-like family in sequence, genomic location, proximity to BGCs, and substrate specificity. This work defines two families of DNA glycosylase for specialized repair of complex genotoxic natural products and generalized repair of a broad range of alkyl-DNA adducts and provides a framework for targeted discovery of new compounds with therapeutic potential.