Yatakemycin biosynthesis requires two deoxyribonucleases for toxin self-resistance

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Yatakemycin (YTM) is a potent DNA-damaging natural product with antimicrobial and antitumor activities.
• The YTM biosynthesis gene cluster includes self-resistance genes, such as ytkR2, which encodes a DNA glycosylase crucial for excising YTM-adenine lesions.

Purpose of the Study:

• To identify and characterize the genes responsible for repairing apurinic/apyrimidinic (AP) sites generated by YTM-induced DNA damage.
• To elucidate the roles of YtkR4 and YtkR5 in the base excision repair (BER) pathway and potential alternative DNA repair mechanisms.

Main Methods:

• Gene identification and characterization (ytkR4, ytkR5).
• Purification and enzymatic activity assays of YtkR4 and YtkR5 (AP endonuclease, 3'-5' exonuclease).
• Analysis of YtkR4 and YtkR5 homologs in natural product gene clusters.

Main Results:

• YtkR4 and YtkR5 were identified as essential for YTM production and encode deoxyribonucleases.
• Purified YtkR4 and YtkR5 demonstrated AP endonuclease activity specific for YtkR2-generated AP sites, supporting a BER pathway.
• YtkR4 and YtkR5 also possess 3'-5' exonuclease activity, with YtkR5 capable of digesting YTM-DNA lesions, suggesting alternative repair mechanisms.
• Homologs of ytkR4 and ytkR5 are frequently found in gene clusters associated with natural product biosynthesis.

Conclusions:

• YtkR4 and YtkR5 are key components of the DNA repair pathway for YTM-induced damage, working in concert with YtkR2.
• The dual enzymatic activities of YtkR4 and YtkR5 highlight their importance in maintaining genomic integrity against genotoxic natural products.
• The co-evolution and clustering of these repair genes suggest a broader role in the biosynthesis and resistance of other DNA-damaging natural products.