Yatakemycin biosynthesis requires two deoxyribonucleases for toxin self-resistance

The highly active natural product yatakemycin (YTM) from Streptomyces sp. TP-A0356 is a potent DNA damaging agent with antimicrobial and antitumor properties. The YTM biosynthesis gene cluster (ytk) contains several toxin self-resistance genes. Of these, ytkR2 encodes a DNA glycosylase that is important for YTM production and host survival by excising lethal YTM-adenine lesions from the genome, presumably initiating a base excision repair (BER) pathway. However, the genes involved in repair of the resulting apurinic/apyrimidinic (AP) site as the second BER step have not been identified. Here, we show that ytkR4 and ytkR5 are essential for YTM production and encode deoxyribonucleases related to other known DNA repair nucleases. Purified YtkR4 and YtkR5 exhibit AP endonuclease activity specific for YtkR2-generated AP sites, providing a basis for BER of the toxic AP intermediate produced from YTM-adenine excision and consistent with co-evolution of ytkR2, ytkR4, and ytkR5. YtkR4 and YtkR5 also exhibit 3'-5' exonuclease activity with differing substrate specificities. The YtkR5 exonuclease is capable of digesting through a YTM-DNA lesion and may represent an alternative repair mechanism to BER. We also show that ytkR4 and ytkR5 homologs are often clustered together in putative gene clusters related to natural product production, consistent with non-redundant roles in repair of other DNA adducts derived from genotoxic natural products.