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National Science Review Aug 2023Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing...
Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine (C) or adenine (A), but no method for guanine (G) or thymine (T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor (gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein (MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 fold. Furthermore, this gGBE exhibited high base editing efficiency (up to 81.2%) and high G-to-T or G-to-C (i.e. G-to-Y) conversion ratio (up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.
PubMed: 37404457
DOI: 10.1093/nsr/nwad143 -
Biochemistry Feb 2020Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA...
Colibactin is a genotoxic gut microbiome metabolite long suspected of playing an etiological role in colorectal cancer. Evidence suggests that colibactin forms DNA interstrand cross-links (ICLs) in eukaryotic cells and activates ICL repair pathways, leading to the production of ICL-dependent DNA double-strand breaks (DSBs). Here we show that colibactin ICLs can evolve directly to DNA DSBs. Using the topology of supercoiled plasmid DNA as a proxy for alkylation adduct stability, we find that colibactin-derived ICLs are unstable toward depurination and elimination of the 3' phosphate. This ICL degradation pathway leads progressively to single strand breaks (SSBs) and subsequently DSBs. The spontaneous conversion of ICLs to DSBs is consistent with the finding that nonhomologous end joining repair-deficient cells are sensitized to colibactin-producing bacteria. The results herein refine our understanding of colibactin-derived DNA damage and underscore the complexities underlying the DSB phenotype.
Topics: Cross-Linking Reagents; DNA; DNA Breaks, Double-Stranded; DNA Breaks, Single-Stranded; DNA Repair; Deoxyribonuclease IV (Phage T4-Induced); Escherichia coli; Peptides; Plasmids; Polyketides
PubMed: 31977191
DOI: 10.1021/acs.biochem.9b01070 -
Biochemistry Feb 2022The G-quadruplex is a noncanonical fold of DNA commonly found at telomeres and within gene promoter regions of the genome. These guanine-rich sequences are highly...
The G-quadruplex is a noncanonical fold of DNA commonly found at telomeres and within gene promoter regions of the genome. These guanine-rich sequences are highly susceptible to damages such as base oxidation and depurination, leading to abasic sites. In the present work, we address whether a vacancy, such as an abasic site, in a G-quadruplex serves as a specific ligand recognition site. When the G-tetrad is all guanines, the vacant (abasic) site is recognized and bound by free guanine nucleobase. However, we aim to understand whether the preference for a specific ligand recognition changes with the presence of a guanine oxidation product 8-oxo-7,8-dihydroguanine (OG) adjacent to the vacancy in the tetrad. Using molecular dynamics simulation, circular dichroism, and nuclear magnetic resonance, we examined the ability for riboflavin to stabilize abasic site-containing G-quadruplex structures. Through structural and free energy binding analysis, we observe riboflavin's ability to stabilize an abasic site-containing G-quadruplex only in the presence of an adjacent OG-modified base. Further, when compared to simulation with the vacancy filled by free guanine, we observe that the free guanine nucleobase is pushed outside of the tetrad by OG to interact with other parts of the structure, including loop residues. These results support the preference of riboflavin over free guanine to fill an OG-adjacent G-quadruplex abasic vacancy.
Topics: Circular Dichroism; DNA; G-Quadruplexes; Guanine; Humans; Magnetic Resonance Spectroscopy; Molecular Dynamics Simulation; Oxidation-Reduction; Promoter Regions, Genetic; Riboflavin; Telomere
PubMed: 35104101
DOI: 10.1021/acs.biochem.1c00598 -
ACS Chemical Biology Dec 2022Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal mG...
Methods for the precise detection and quantification of RNA modifications are critical to uncover functional roles of diverse RNA modifications. The internal mG modification in mammalian cytoplasmic tRNAs is known to affect tRNA function and impact embryonic stem cell self-renewal, tumorigenesis, cancer progression, and other cellular processes. Here, we introduce mG-quant-seq, a quantitative method that accurately detects internal mG sites in human cytoplasmic tRNAs at single-base resolution. The efficient chemical reduction and mild depurination can almost completely convert internal mG sites into RNA abasic sites (AP sites). We demonstrate that RNA abasic sites induce a mixed variation pattern during reverse transcription, including G → A or C or T mutations as well as deletions. We calculated the total variation ratio to quantify the mG modification fraction at each methylated site. The calibration curves of all relevant motif contexts allow us to more quantitatively determine the mG methylation level. We detected internal mG sites in 22 human cytoplasmic tRNAs from HeLa and HEK293T cells and successfully estimated the corresponding mG methylation stoichiometry. mG-quant-seq could be applied to monitor the tRNA mG methylation level change in diverse biological processes.
Topics: Animals; Humans; RNA; HEK293 Cells; Guanosine; RNA, Transfer; Mammals
PubMed: 36398936
DOI: 10.1021/acschembio.2c00792 -
Phytochemistry Oct 2022Ribosome inactivating proteins (RIPs) are rRNA N-glycosylases (EC 3.2.2.22) best known for hydrolyzing an adenine base from the conserved sarcin/ricin loop of ribosomal... (Review)
Review
Ribosome inactivating proteins (RIPs) are rRNA N-glycosylases (EC 3.2.2.22) best known for hydrolyzing an adenine base from the conserved sarcin/ricin loop of ribosomal RNA. Protein translation is inhibited by ribosome depurination; therefore, RIPs are generally considered toxic to cells. The expression of some RIPs is upregulated by biotic and abiotic stress, though the connection between RNA depurination and defense response is not well understood. Despite their prevalence in approximately one-third of flowering plant orders, our knowledge of RIPs stems primarily from biochemical analyses of individuals or genomics-scale analyses of small datasets from a limited number of species. Here, we performed an unbiased search for proteins with RIP domains and identified several-fold more RIPs than previously known - more than 800 from 120 species, many with novel associated domains and physicochemical characteristics. Based on protein domain configuration, we established 15 distinct groups, suggesting diverse functionality. Surprisingly, most of these RIPs lacked a signal peptide, indicating they may be localized to the nucleocytoplasm of cells, raising questions regarding their toxicity against conspecific ribosomes. Our phylogenetic analysis significantly extends previous models for RIP evolution in plants, predicting an original single-domain RIP that later evolved to acquire a signal peptide and different protein domains. We show that RIPs are distributed throughout 21 plant orders with many species maintaining genes for more than one RIP group. Our analyses provide the foundation for further characterization of these new RIP types, to understand how these enzymes function in plants.
Topics: Phylogeny; Plant Proteins; Protein Sorting Signals; RNA, Ribosomal; Ribosome Inactivating Proteins; Ribosomes
PubMed: 35934106
DOI: 10.1016/j.phytochem.2022.113337 -
Cell Biochemistry and Function Jan 2022DNA repair is an important pathway for the protection of DNA molecules from destruction. DNA damage can be produced by oxidative reactive nitrogen or oxygen species,... (Review)
Review
DNA repair is an important pathway for the protection of DNA molecules from destruction. DNA damage can be produced by oxidative reactive nitrogen or oxygen species, irritation, alkylating agents, depurination and depyrimidination; in this regard, DNA repair pathways can neutralize the negative effects of these factors. Melatonin is a hormone secreted from the pineal gland with an antioxidant effect by binding to oxidative factors. In addition, the effect of melatonin on DNA repair pathways has been proven by the literature. DNA repair is carried out by several mechanisms, of which homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) are of great importance. Because of the importance of DNA repair in DNA integrity and the anticancer effect of this pathway, we presented the effect of melatonin on DNA repair factors regarding previous studies conducted in this area.
Topics: DNA; DNA Damage; DNA End-Joining Repair; DNA Repair; Melatonin
PubMed: 34672014
DOI: 10.1002/cbf.3672 -
Toxins Feb 2022Tian Hua Fen, a herbal powder extract that contains trichosanthin (TCS), was used as an abortifacient in traditional Chinese medicine. In 1972, TCS was purified to... (Review)
Review
Tian Hua Fen, a herbal powder extract that contains trichosanthin (TCS), was used as an abortifacient in traditional Chinese medicine. In 1972, TCS was purified to alleviate the side effects. Because of its clinical applications, TCS became one of the most active research areas in the 1960s to the 1980s in China. These include obtaining the sequence information in the 1980s and the crystal structure in 1995. The replication block of TCS on human immunodeficiency virus in lymphocytes and macrophages was found in 1989 and started a new chapter of its development. Clinical studies were subsequently conducted. TCS was also found to have the potential for gastric and colorectal cancer treatment. Studies on its mechanism showed TCS acts as an rRNA -glycosylase (EC 3.2.2.22) by hydrolyzing and depurinating A-4324 in α-sarcin/ricin loop on 28S rRNA of rat ribosome. Its interaction with acidic ribosomal stalk proteins was revealed in 2007, and its trafficking in mammalian cells was elucidated in the 2000s. The adverse drug reactions, such as inducing immune responses, short plasma half-life, and non-specificity, somehow became the obstacles to its usage. Immunotoxins, sequence modification, or coupling with polyethylene glycerol and dextran were developed to improve the pharmacological properties. TCS has nicely shown the scientific basis of traditional Chinese medicine and how its research and development have expanded the knowledge and applications of ribosome-inactivating proteins.
Topics: Animals; Mammals; Rats; Research; Ribosomal Proteins; Ribosomes; Saporins; Trichosanthin
PubMed: 35324675
DOI: 10.3390/toxins14030178 -
Toxins Mar 2020Ribosome-inactivating proteins (RIPs) are N-glycosidases, which depurinate a specific adenine residue in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. This loop... (Review)
Review
Ribosome-inactivating proteins (RIPs) are N-glycosidases, which depurinate a specific adenine residue in the conserved α-sarcin/ricin loop (α-SRL) of rRNA. This loop is important for anchoring elongation factor (EF-G for prokaryote or eEF2 for eukaryote) in mRNA translocation. Translation is inhibited after the attack. RIPs therefore may have been applied for anti-cancer, and anti-virus and other therapeutic applications. The main obstacles of treatment with RIPs include short plasma half-life, non-selective cytotoxicity and antigenicity. This review focuses on the strategies used to improve the pharmacological properties of RIPs on human immunodeficiency virus (HIV) and cancers. Coupling with polyethylene glycol (PEG) increases plasma time and reduces antigenicity. RIPs conjugated with antibodies to form immunotoxins increase the selective toxicity to target cells. The prospects for future development on the engineering of RIPs for improving their pharmacological properties are also discussed.
Topics: Animals; Anti-HIV Agents; Antineoplastic Agents; Dextrans; Humans; Polyethylene Glycols; Protein Engineering; Ribosome Inactivating Proteins
PubMed: 32182799
DOI: 10.3390/toxins12030167 -
Frontiers in Immunology 2020The integrity of the genome is under constant threat of environmental and endogenous agents that cause DNA damage. Endogenous damage is particularly pervasive, occurring... (Review)
Review
The integrity of the genome is under constant threat of environmental and endogenous agents that cause DNA damage. Endogenous damage is particularly pervasive, occurring at an estimated rate of 10,000-30,000 per cell/per day, and mostly involves chemical DNA base lesions caused by oxidation, depurination, alkylation, and deamination. The base excision repair (BER) pathway is primary responsible for removing and repairing these small base lesions that would otherwise lead to mutations or DNA breaks during replication. Next to preventing DNA mutations and damage, the BER pathway is also involved in mutagenic processes in B cells during immunoglobulin (Ig) class switch recombination (CSR) and somatic hypermutation (SHM), which are instigated by uracil (U) lesions derived from activation-induced cytidine deaminase (AID) activity. BER is required for the processing of AID-induced lesions into DNA double strand breaks (DSB) that are required for CSR, and is of pivotal importance for determining the mutagenic outcome of uracil lesions during SHM. Although uracils are generally efficiently repaired by error-free BER, this process is surprisingly error-prone at the loci in proliferating B cells. Breakdown of this high-fidelity process outside of the loci has been linked to mutations observed in B-cell tumors and DNA breaks and chromosomal translocations in activated B cells. Next to its role in preventing cancer, BER has also been implicated in immune tolerance. Several defects in BER components have been associated with autoimmune diseases, and animal models have shown that BER defects can cause autoimmunity in a B-cell intrinsic and extrinsic fashion. In this review we discuss the contribution of BER to genomic integrity in the context of immune receptor diversification, cancer and autoimmune diseases.
Topics: Animals; B-Lymphocytes; DNA; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; Humans; Immune System; Immunoglobulin Class Switching; Somatic Hypermutation, Immunoglobulin
PubMed: 32547565
DOI: 10.3389/fimmu.2020.01084 -
Angewandte Chemie (International Ed. in... Sep 2021DNA-encoded compound libraries are a widely used small molecule screening technology. One important aim in library design is the coverage of chemical space through...
DNA-encoded compound libraries are a widely used small molecule screening technology. One important aim in library design is the coverage of chemical space through structurally diverse molecules. Yet, the chemical reactivity of native DNA barcodes limits the toolbox of reactions for library design. Substituting the chemically vulnerable purines by 7-deazaadenine, which exhibits tautomerization stability similar to natural adenine with respect to the formation of stable Watson-Crick pairs, yielded ligation-competent, amplifiable, and readable DNA barcodes for encoded chemistry with enhanced stability against protic acid- and metal ion-promoted depurination. The barcode stability allowed for straightforward translation of 16 exemplary reactions that included isocyanide multicomponent reactions, acid-promoted Pictet-Spengler and Biginelli reactions, and metal-promoted pyrazole syntheses on controlled pore glass-coupled barcodes for diverse DEL design. The Boc protective group of reaction products offered a convenient handle for encoded compound purification.
PubMed: 34153170
DOI: 10.1002/anie.202104348