独特的DNA修复酶,提供对治疗重要的自我抗性,基因毒性天然产物已在细菌生物合成基因簇(BGC)中发现。其中,DNA糖基化酶AlkZ是生产氮杂霉素B所必需的,属于未表征蛋白质的HTH_42超家族。尽管它们广泛存在于抗生素生产者和病原体中,这些蛋白质在生产其他天然产物中的作用尚不清楚。这里,我们确定了来自链霉菌的所有HTH_42蛋白的进化关系和基因组分布,并使用基于抗性的基因组挖掘方法来鉴定与已知和未表征的BGC相关的同源物。我们发现AlkZ-like(AZL)蛋白构成了一个独特的HTH_42亚家族,在BGC中高度富集,序列可变,表明每个人都进化来保护自己免受特定的次级代谢产物的侵害。作为对该方法的验证,我们发现AZL蛋白,HedH4,与烷化剂hedamycin的生物合成相关,切除hedamycin-DNA加合物,并提供对细胞中天然产物的抗性。我们还确定了第二个,系统发育和功能上不同的亚家族,其蛋白质从未与BGC相关,在序列和基因组邻域方面高度保守,修复与特定天然产物无关的DNA损伤。这项工作描述了两个相关的DNA修复酶家族-一个对复杂的烷基DNA损伤具有特异性,并且涉及对抗菌剂的自我抗性,另一个可能涉及针对一系列基因毒素的保护-并为有针对性地发现具有治疗潜力的新基因毒性化合物提供了框架。重要性细菌是次级代谢产物的丰富来源,包括具有抗肿瘤/抗生素特性的DNA损伤基因毒素。虽然链霉菌产生不同数量的治疗基因毒素,缺乏有针对性地发现产生DNA损伤剂的生物合成基因簇(BGC)的努力。此外,抗毒素基因的研究已经落后于我们对那些参与天然产物合成的基因的理解。这里,我们使用氮杂霉素B抗性DNA糖基化酶AlkZ,通过基于抗性的基因组挖掘,鉴定了超过70个未表征的BGC,这些BGC产生潜在的新型基因毒素.我们通过表征河达霉素BGC中一种AlkZ直系同源物的酶活性和细胞抗性来验证我们的分析,一种有效的DNA烷化剂。此外,我们发现一秒钟,与大肠杆菌YcaQ相关的系统发育上不同的蛋白质家族,一种能够解开链间DNA交联的DNA糖基化酶,在序列上不同于AlkZ类家族,基因组位置,接近BGC,和底物特异性。这项工作定义了DNA糖基化酶的两个家族,用于复杂的基因毒性天然产物的专门修复和广泛的烷基-DNA加合物的广泛修复,并为靶向发现具有治疗潜力的新化合物提供了框架。
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.