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

   https://doi.org/10.1128/mbio.03297-21  

   Bradley, Noah P.; Wahl, Katherine L.; Steenwyk, Jacob L.; Rokas, Antonis; Eichman, Brandt F. (April 2022, mBio)

   Simmons, Lyle A.; Bush, Karen (Ed.)

   ABSTRACT 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. 

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2. FSHing for DNA Damage: Key Features of MutY Detection of 8-Oxoguanine:Adenine Mismatches

   https://doi.org/10.1021/acs.accounts.3c00759  

   Majumdar, Chandrima; Demir, Merve; Merrill, Steven R; Hashemian, Mohammad; David, Sheila S (April 2024, Accounts of Chemical Research)

   Base excision repair (BER) enzymes are genomic superheroes that stealthly and accurately identify and remove chemically modified DNA bases. DNA base modifications erode the informational content of DNA and underly many disease phenotypes, most conspicuously, cancer. The “OG” of oxidative base damage, 8-oxo-7,8-dihydroguanine (OG), is particularly insidious due to its miscoding ability that leads to formation of rare pro-mutagenic OG:A mismatches. Thwarting mutagenesis relies on capture of OG:A mismatches prior to DNA replication and removal of the mis-inserted adenine by MutY glycosylases to initiate BER. The threat of OG and the importance of its repair is underscored by the association between inherited dysfunctional variants of the MutY human homolog MUTYH and colorectal cancer, known as MUTYH-associated polyposis (MAP). Our functional studies of the two founder MUTYH variants revealed that both have a compromised activity and reduced affinity for OG:A mismatches. Indeed, these studies underscored the challenge of recognition of OG:A mismatches that are only subtlely structurally different than T:A base-pairs. Since the original discovery of MAP, many MUTYH variants have been reported, with most considered “variants of uncertain significance.” To reveal features associated with damage recognition and adenine excision by MutY and MUTYH, we have developed a multi-pronged chemical biology approach combining enzyme kinetics, X-ray crystallography, single-molecule visualization, and cellular repair assays. In this review, we highlight recent work in our laboratory where we defined MutY structure-activity relationship (SAR) studies using synthetic analogs of OG and A in cellular and in vitro assays. Our studies revealed the 2-amino group of OG as the key distinguishing feature of OG:A mismatches. Indeed, the unique position of the 2-amino group in the major groove of OGsyn:Aanti mismatches provides a means for its rapid detection among a large excess of highly abundant and structurally similar canonical base pairs. Furthermore, site-directed mutagenesis and structural analysis showed that a conserved C-terminal domain β-hairpin “FSH'' loop is critical for OG recognition with the “His” serving as the lesion detector. Notably, MUTYH variants located within and near the FSH loop have been associated with different forms of cancer. Uncovering the role(s) of this loop in lesion recognition provided a detailed understanding of the search and repair process of MutY. Such insights are also useful to identify mutational hotspots and pathogenic variants, that may improve the ability of physicians to diagnose the likelihood of diseases onset and prognosis. The critical importance of the “FSH” loop in lesion detection suggests that it may serve as a unique locus for targeting of probes or inhibitors of MutY/MUTYH to provide new chemical biology tools, and avenues for therapeutic development. 

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3. The Landscape of Phenotypic and Transcriptional Responses to Ciprofloxacin in Acinetobacter baumannii: Acquired Resistance Alleles Modulate Drug-Induced SOS Response and Prophage Replication

   https://doi.org/10.1128/mBio.01127-19  

   Geisinger, Edward; Vargas-Cuebas, Germán; Mortman, Nadav J.; Syal, Sapna; Dai, Yunfei; Wainwright, Elizabeth L.; Lazinski, David; Wood, Stephen; Zhu, Zeyu; Anthony, Jon; et al (June 2019, mBio)

   Miller, Samuel I. (Ed.)

   ABSTRACT The emergence of fluoroquinolone resistance in nosocomial pathogens has restricted the clinical efficacy of this antibiotic class. In Acinetobacter baumannii , the majority of clinical isolates now show high-level resistance due to mutations in gyrA (DNA gyrase) and parC (topoisomerase IV [topo IV]). To investigate the molecular basis for fluoroquinolone resistance, an exhaustive mutation analysis was performed in both drug-sensitive and -resistant strains to identify loci that alter ciprofloxacin sensitivity. To this end, parallel fitness tests of over 60,000 unique insertion mutations were performed in strains with various alleles in genes encoding the drug targets. The spectra of mutations that altered drug sensitivity were found to be similar in the drug-sensitive and gyrA parC double-mutant backgrounds, having resistance alleles in both genes. In contrast, the introduction of a single gyrA resistance allele, resulting in preferential poisoning of topo IV by ciprofloxacin, led to extreme alterations in the insertion mutation fitness landscape. The distinguishing feature of preferential topo IV poisoning was enhanced induction of DNA synthesis in the region of two endogenous prophages, with DNA synthesis associated with excision and circularization of the phages. Induction of the selective DNA synthesis in the gyrA background was also linked to heightened prophage gene transcription and enhanced activation of the mutagenic SOS response relative to that observed in either the wild-type (WT) or gyrA parC double mutant. Therefore, the accumulation of mutations that result in the stepwise evolution of high ciprofloxacin resistance is tightly connected to modulation of the SOS response and endogenous prophage DNA synthesis. IMPORTANCE Fluoroquinolones have been extremely successful antibiotics due to their ability to target multiple bacterial enzymes critical to DNA replication, the topoisomerases DNA gyrase and topo IV. Unfortunately, mutations lowering drug affinity for both enzymes are now widespread, rendering these drugs ineffective for many pathogens. To undermine this form of resistance, we examined how bacteria with target alterations differentially cope with fluoroquinolone exposures. We studied this problem in the nosocomial pathogen A. baumannii , which causes drug-resistant life-threatening infections. Employing genome-wide approaches, we uncovered numerous pathways that could be exploited to raise fluoroquinolone sensitivity independently of target alteration. Remarkably, fluoroquinolone targeting of topo IV in specific mutants caused dramatic hyperinduction of prophage replication and enhanced the mutagenic DNA damage response, but these responses were muted in strains with DNA gyrase as the primary target. This work demonstrates that resistance evolution via target modification can profoundly modulate the antibiotic stress response, revealing potential resistance-associated liabilities. 

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4. Uracil-DNA glycosylase efficiency is modulated by substrate rigidity

   https://doi.org/10.1038/s41598-023-30620-0  

   Orndorff, Paul B.; Poddar, Souvik; Owens, Aerial M.; Kumari, Nikita; Ugaz, Bryan T.; Amin, Samrat; Van Horn, Wade D.; van der Vaart, Arjan; Levitus, Marcia (December 2023, Scientific Reports)

   Abstract Uracil DNA-glycosylase (UNG) is a DNA repair enzyme that removes the highly mutagenic uracil lesion from DNA using a base flipping mechanism. Although this enzyme has evolved to remove uracil from diverse sequence contexts, UNG excision efficiency depends on DNA sequence. To provide the molecular basis for rationalizing UNG substrate preferences, we used time-resolved fluorescence spectroscopy, NMR imino proton exchange measurements, and molecular dynamics simulations to measure UNG specificity constants ( k cat / K M ) and DNA flexibilities for DNA substrates containing central AUT, TUA, AUA, and TUT motifs. Our study shows that UNG efficiency is dictated by the intrinsic deformability around the lesion, establishes a direct relationship between substrate flexibility modes and UNG efficiency, and shows that bases immediately adjacent to the uracil are allosterically coupled and have the greatest impact on substrate flexibility and UNG activity. The finding that substrate flexibility controls UNG efficiency is likely significant for other repair enzymes and has major implications for the understanding of mutation hotspot genesis, molecular evolution, and base editing. 

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5. Structures and activity for Geobacillus stearothermophilus MutY Glu43 substitution variants: Implications for evolution of an adenine DNA glycosylase

   https://doi.org/10.1021/scimeetings.5c11452  

   Horvath, MP; Russelburg, LP; Demir, M; Cedeno, K; David, SS (August 2025, ACS SciMeetings)

   Background: MutY initiated base excision repair (BER) protects against mutations that otherwise result from oxidative damage to guanine. The 8-oxo-7,8-dihydro-guanine lesion (OG) pairs equally well with C or A, a source of ambiguity that explains high rates of GC → TA mutations for biallelic defects in the gene encoding MUTYH in humans. MutY/MUTYH intercepts the A:OG lesion and removes the chemically correct but informationally defective adenine nucleobase. A catalytic Glu – Glu43 in Geobacillus stearothermophilus MutY (Gs MutY); Glu37 in Escherichia coli MutY (Ec MutY); Glu134 in human MUTYH – is a defining chemical motif for A:OG adenine DNA glycosylases that is not found in other helix-hairpin-helix family members. While the importance of Glu for MutY’s catalytic mechanism is well understood, its origin is unknown. Discovery: Here we tested the structural and kinetic consequences of Glu replacement and found an alternate, slower mechanism, that yields a different stereoisomer for the AP site product. The impact on catalytic rate was consistent removal of acid/base catalysis and retention of transition state stabilization. High-resolution structures for E43Q and E43S substitution variants of Gs MutY show substrate disengagement and the enzyme-generated AP product in its alpha-anomer configuration. These results suggest that acquisition of Glu was a key innovation for emergence of the MutY/MUTYH lineage from an ancestral DNA glycosylase. 

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