Cell-cycle checkpoints and DNA repair processes protect organisms from potentially lethal mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low Guanine–Cytosine (GC) content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18/21 known species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, a faster-evolving lineage (FEL), which began diversifying approximately 87 million years ago (mya), and a slower-evolving lineage (SEL), which began diversifying approximately 54 mya. Remarkably, both lineages lost genes associated with the cell cycle and genome integrity, but these losses were greater in the FEL. E.g., all species lost the cell-cycle regulator WHIskey 5 ( WHI5 ), and the FEL lost components of the spindle checkpoint pathway (e.g., Mitotic Arrest-Deficient 1 [ MAD1 ], Mitotic Arrest-Deficient 2 [ MAD2 ]) and DNA-damage–checkpoint pathway (e.g., Mitosis Entry Checkpoint 3 [ MEC3 ], RADiation sensitive 9 [ RAD9 ]). Similarly, both lineages lost genes involved in DNA repair pathways, including the DNA glycosylase gene 3-MethylAdenine DNA Glycosylase 1 ( MAG1 ), which is part of the base-excision repair pathway, and the DNA photolyase gene PHotoreactivation Repair deficient 1 ( PHR1 ), which is involved in pyrimidine dimer repair. Strikingly, the FEL lost 33 additional genes, including polymerases (i.e., POLymerase 4 [ POL4 ] and POL32 ) and telomere-associated genes (e.g., Repressor/activator site binding protein-Interacting Factor 1 [ RIF1 ], Replication Factor A 3 [ RFA3 ], Cell Division Cycle 13 [ CDC13 ], Pbp1p Binding Protein [ PBP2 ]). Echoing these losses, molecular evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, and higher fractions of mutations associated with the common endogenously damaged base, 8-oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and thrived, despite lacking many otherwise highly conserved cell-cycle and genome integrity genes and pathways, and may represent a novel, to our knowledge, system for studying cellular life without them.
DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a β-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.
33Cell cycle checkpoints and DNA repair processes protect organisms from potentially lethal 34 mutational damage. Compared to other budding yeasts in the subphylum Saccharomycotina, we 35 noticed that a lineage in the genus Hanseniaspora exhibited very high evolutionary rates, low 36 GC content, small genome sizes, and lower gene numbers. To better understand Hanseniaspora 37 evolution, we analyzed 25 genomes, including 11 newly sequenced, representing 18 / 21 known 38 species in the genus. Our phylogenomic analyses identify two Hanseniaspora lineages, the fast-39 evolving lineage (FEL), which began diversifying ~87 million years ago (mya), and the slow-40 evolving lineage (SEL), which began diversifying ~54 mya. Remarkably, both lineages lost 41 genes associated with the cell cycle and genome integrity, but these losses were greater in the 42 FEL. For example, all species lost the cell cycle regulator WHI5, and the FEL lost components of 43 the spindle checkpoint pathway (e.g., MAD1, MAD2) and DNA damage checkpoint pathway 44 (e.g., MEC3, RAD9). Similarly, both lineages lost genes involved in DNA repair pathways, 45including the DNA glycosylase gene MAG1, which is part of the base excision repair pathway, 46 and the DNA photolyase gene PHR1, which is involved in pyrimidine dimer repair. Strikingly, 47 the FEL lost 33 additional genes, including polymerases (i.e., POL4 and POL32) and telomere-48 associated genes (e.g., RIF1, RFA3, CDC13, PBP2). Echoing these losses, molecular 49 evolutionary analyses reveal that, compared to the SEL, the FEL stem lineage underwent a burst 50 of accelerated evolution, which resulted in greater mutational loads, homopolymer instabilities, 51 and higher fractions of mutations associated with the common endogenously damaged base, 8-52 oxoguanine. We conclude that Hanseniaspora is an ancient lineage that has diversified and 53 thrived, despite lacking many otherwise highly conserved cell cycle and genome integrity genes 54 and pathways, and may represent a novel system for studying cellular life without them. 55 Introduction 56Genome maintenance is largely attributed to the fidelity of cell cycle checkpoints, DNA repair 57 pathways, and their interaction [1]. Dysregulation of these processes often leads to the loss of 58 genomic integrity [2] and hypermutation, or the acceleration of mutation rates [3]. For example, 59 improper control of cell cycle and DNA repair processes can lead to 10-to 100-fold increases in 60 mutation rate [4]. Furthermore, deletions of single genes can have profound effects on genome 61 stability. For example, the deletion of MEC3, which is involved in sensing DNA damage in the 62 G1 and G2/M cell cycle phases, can lead to a 54-fold increase in the gross chromosomal 63 rearrangement rate [5]. Similarly, nonsense mutations in mismatch repair proteins account for the 64 emergence of hypermutator strains in the yeast pathogens Cryptococcus deuterogattii [6] and 65Cryptococcus neoformans [7,8]. Due to their importance in ensuring genomic integrity, most 66 genome...
Two families of DNA glycosylases (YtkR2/AlkD, AlkZ/YcaQ) have been found to remove bulky and crosslinking DNA adducts produced by bacterial natural products. Whether DNA glycosylases eliminate other types of damage formed by structurally diverse antibiotics is unknown. Here, we identify four DNA glycosylases—TxnU2, TxnU4, LldU1 and LldU5—important for biosynthesis of the aromatic polyketide antibiotics trioxacarcin A (TXNA) and LL-D49194 (LLD), and show that the enzymes provide self-resistance to the producing strains by excising the intercalated guanine adducts of TXNA and LLD. These enzymes are highly specific for TXNA/LLD-DNA lesions and have no activity toward other, less stable alkylguanines as previously described for YtkR2/AlkD and AlkZ/YcaQ. Similarly, TXNA-DNA adducts are not excised by other alkylpurine DNA glycosylases. TxnU4 and LldU1 possess unique active site motifs that provide an explanation for their tight substrate specificity. Moreover, we show that abasic (AP) sites generated from TxnU4 excision of intercalated TXNA-DNA adducts are incised by AP endonuclease less efficiently than those formed by 7mG excision. This work characterizes a distinct class of DNA glycosylase acting on intercalated DNA adducts and furthers our understanding of specific DNA repair self-resistance activities within antibiotic producers of structurally diverse, highly functionalized DNA damaging agents.
Interstrand DNA crosslinks (ICLs) are a toxic form of DNA damage that block DNA replication and transcription by tethering the opposing strands of DNA. ICL repair requires unhooking of the tethered strands by either nuclease incision of the DNA backbone or glycosylase cleavage of the crosslinked nucleotide. In bacteria, glycosylase-mediated ICL unhooking was described in Streptomyces as a means of self-resistance to the genotoxic natural product azinomycin B. The mechanistic details and general utility of glycosylase-mediated ICL repair in other bacteria are unknown. Here, we identify the uncharacterized Escherichia coli protein YcaQ as an ICL repair glycosylase that protects cells against the toxicity of crosslinking agents. YcaQ unhooks both sides of symmetric and asymmetric ICLs in vitro , and loss or overexpression of ycaQ sensitizes E. coli to the nitrogen mustard mechlorethamine. Comparison of YcaQ and UvrA-mediated ICL resistance mechanisms establishes base excision as an alternate ICL repair pathway in bacteria.
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.
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