Yatakemycin (YTM) is an extraordinarily toxic DNA alkylating agent with potent antimicrobial and antitumor properties and the most recent addition to the CC-1065 and duocarmycin family of natural products. While bulky DNA lesions the size of those produced by YTM are normally removed from the genome by the nucleotide excision repair (NER) pathway, YTM adducts are also a substrate for the bacterial DNA glycosylases AlkD and YtkR2, unexpectedly implicating base excision repair (BER) in their elimination. The reason for the extreme toxicity of these lesions and the molecular basis for how they are eliminated by BER have been unclear. Here, we describe the structural and biochemical properties of YTM adducts responsible for their toxicity, and define the mechanism by which they are excised by AlkD. These findings delineate an alternative strategy for repair of bulky DNA damage and establish the cellular utility of this pathway relative to that of NER.
DNA glycosylases preserve genome integrity and define the specificity of the base excision repair pathway for discreet, detrimental modifications, and thus, the mechanisms by which glycosylases locate DNA damage are of particular interest. Bacterial AlkC and AlkD are specific for cationic alkylated nucleobases and have a distinctive HEAT-like repeat (HLR) fold. AlkD uses a unique non-base-flipping mechanism that enables excision of bulky lesions more commonly associated with nucleotide excision repair. In contrast, AlkC has a much narrower specificity for small lesions, principally N3-methyladenine (3mA). Here, we describe how AlkC selects for and excises 3mA using a non-base-flipping strategy distinct from that of AlkD. A crystal structure resembling a catalytic intermediate complex shows how AlkC uses unique HLR and immunoglobulin-like domains to induce a sharp kink in the DNA, exposing the damaged nucleobase to active site residues that project into the DNA This active site can accommodate and excise N3-methylcytosine (3mC) and N1-methyladenine (1mA), which are also repaired by AlkB-catalyzed oxidative demethylation, providing a potential alternative mechanism for repair of these lesions in bacteria.
DNA glycosylases are important repair enzymes that eliminate a diverse array of aberrant nucleobases from the genomes of all organisms. Individual bacterial species often contain multiple paralogs of a particular glycosylase, yet the molecular and functional distinctions between these paralogs are not well understood. The recently discovered HEAT-like repeat (HLR) DNA glycosylases are distributed across all domains of life and are distinct in their specificity for cationic alkylpurines and mechanism of damage recognition. Here, we describe a number of phylogenetically diverse bacterial species with two orthologs of the HLR DNA glycosylase AlkD. One ortholog, which we designate AlkD2, is substantially less conserved. The crystal structure of Streptococcus mutans AlkD2 is remarkably similar to AlkD but lacks the only helix present in AlkD that penetrates the DNA minor groove. We show that AlkD2 possesses only weak DNA binding affinity and lacks alkylpurine excision activity. Mutational analysis of residues along this DNA binding helix in AlkD substantially reduced binding affinity for damaged DNA, for the first time revealing the importance of this structural motif for damage recognition by HLR glycosylases.
DNA glycosylases remove aberrant DNA nucleobases as the first enzymatic step of the base excision repair (BER) pathway. The alkyl‐DNA glycosylases AlkC and AlkD adopt a unique structure based on α‐helical HEAT repeats. Both enzymes identify and excise their substrates without a base‐flipping mechanism used by other glycosylases and nucleic acid processing proteins to access nucleobases that are otherwise stacked inside the double‐helix. Consequently, these glycosylases act on a variety of cationic nucleobase modifications, including bulky adducts, not previously associated with BER. The related non‐enzymatic HEAT‐like repeat (HLR) proteins, AlkD2, and AlkF, have unique nucleic acid binding properties that expand the functions of this relatively new protein superfamily beyond DNA repair. Here, we review the phylogeny, biochemistry, and structures of the HLR proteins, which have helped broaden our understanding of the mechanisms by which DNA glycosylases locate and excise chemically modified DNA nucleobases.
Threats to genomic integrity are mitigated by DNA glycosylases, which initiate the base excision repair pathway by locating and excising aberrant nucleobases. A hallmark of these and other DNA repair enzymes is their use of base flipping to sequester modified nucleotides from the DNA helix and into an active site pocket. Consequently, base flipping is generally regarded as an essential aspect of lesion recognition and a necessary precursor to base excision. We recently described the first DNA glycosylase mechanism that does not require base flipping for either binding or catalysis1. The DNA glycosylase AlkD recognizes aberrant base pairs through contacts with the phosphoribose backbone, while the damaged nucleobase remains stacked in the DNA duplex, and and uses catalytic CH–π and charge–dipole interactions to preferentially stabilize the transition state. We now show through a combination of crystallographic, biochemical, biophysical, and cellular techniques how this unique mechanism enables AlkD to repair large adducts formed by yatakemycin (Fig. 1), a member of the duocarmycin and CC‐1065 family of antimicrobial and antitumor natural products. Bulky adducts of this, or any type, are not excised by DNA glycosylases that use a traditional base‐flipping mechanism. Hence, these findings represent a new paradigm for DNA repair and provide insights into damage recognition and base excision.Support or Funding InformationThis work was funded by the National Science Foundation (MCB‐1122098 and MCB‐1517695) and the National Institutes of Health (R01ES019625).
BackgroundUndifferentiated pleomorphic sarcoma (UPS) in oral-maxillary area is rarely reported. Herein, we aimed to investigate the clinical characteristics, treatment strategies, prognosis, and molecular features of the oral-maxillary UPS. MethodsRetrospectively, we reviewed the UPS patients who were diagnosed and treated in our department. The medical histories, imaging features, histopathological characteristics, treatment strategies, clinical outcomes were summarized and analyzed. Besides, the molecular features were demonstrated by whole exonic sequencing. ResultsTotally, 10 cases with primary oral-maxillary UPS were included. The rapidly progressive UPS can easily develop at advanced and life-threatening stage, especially concerning the complex anatomical structures and spaces in the oral-maxillary area. The finial diagnosis for UPS greatly depends on histological findings and immunohistochemistry under the exclusion of all the possible differential diagnosis. Retrospectively, the treatment strategies for the involved cases still referred to those of oral squamous cell carcinoma. Statistically, the median overall survival (OS) for all the included cases was 7.75 months (range: 5-17 months). Comparatively, 3 cases had improved OS (median survival: 17 months, range: 17-18 months) experienced PR/SD with neoadjuvant chemotherapy (anlotinib). Cancer driver genes detection revealed GBP4 as a candidate driver gene for the oral-maxillary UPS. Additionally, a missense mutation in gene PIK3CA (p.E545K) was also identified. ConclusionOur findings can greatly expand the knowledge about the oral-maxillary UPS, and provide molecular evidences to improve the therapeutic options for the oral-maxillary UPS. Further studies are warranted to validate our discoveries on the oral-maxillary UPS.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.