Yatakemycin biosynthesis requires two deoxyribonucleases for toxin self-resistance

The highly active natural product yatakemycin (YTM) from <i>Streptomyces</i> sp. TP-A0356 is a potent DNA damaging agent with antimicrobial and antitumor properties. The YTM biosynthesis gene cluster (<i>ytk</i>) contains several toxin self-resistance genes. Of these, <i>ytkR2</i> 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 <i>ytkR4</i> and <i>ytkR5</i> 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 <i>ytkR2</i>, <i>ytkR4</i>, and <i>ytkR5</i>. 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 <i>ytkR4</i> and <i>ytkR5</i> 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.