Base excision repair system targeting DNA adducts of trioxacarcin/LL-D49194 antibiotics for self-resistance

Abstract

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.

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

“…S. sahachiroi AlkZ, S. bottropensis TxnU2 and TxnU4, and S. vinaceusdrappus LldU1 and LldU5, like HedH4, are found in BGCs that produce bulky N 7-alkyl- and intercalating DNA adducts ( Fig. 3A ), and each is specific for their cognate toxin ( 31 , 56 ). Compared to HedH4, which excises 100% of the HED-guanine from DNA, none of the 10 alkylpurine DNA glycosylases tested showed any appreciable activity for HED-DNA after 1 h ( Fig.…”

“…HedH4 was also unable to hydrolyze TXNA-guanosine, a substrate for TxnU4 from the TXNA BGC ( Fig. 4F ) ( 56 ). We also tested the ability of HedH4 to unhook ICLs derived from AZB ( Fig.…”

“…We show that the AZL protein, HedH4, within the HED cluster specifically excises HED-DNA adducts and improves viability of cells grown in the presence of the compound. In a separate study, we recently found that the two paralogs present in TXN and LLD clusters (TxnU2, TxnU4, LldU1, and LldU5) are self-resistance glycosylases for these compounds ( 56 ). Thus, together with the previous example from the AZB BGC ( 28 , 31 ), there is now mounting evidence that AZL family genes have evolved largely as DNA repair self-resistance proteins against a variety of natural products.…”

“…It is likely that the HedH4-generated HED-guanine moiety remains intercalated at the AP site and requires a specialized AP endonuclease for repair. Indeed, we recently found that the excised guanine adduct of the related, intercalating natural product TXNA is a poor substrate for E. coli EndoIV ( 56 ). More pertinent to HED biosynthesis, the producing organism S. griseoruber contains two copies each of ExoIII- and EndoIV-like AP endonucleases that may have evolved to incise HED AP sites, although none are located in the hed BGC.…”

“…HED ( Streptomyces griseoruber ATCC 23919) was obtained from the National Cancer Institute’s Developmental Therapeutic Program (NCI DTP) Open Compound Repository (NSC 70929). Trioxacarcin A (TXNA) was isolated from Streptomyces bottropensis NRRL 12051 as described previously ( 56 ). AZB was prepared from organic extract of Streptomyces sahachiroi (ATCC 33158) as in reference 31 .…”