The 8-oxoguanine DNA glycosylase-synaptotagmin 7 pathway increases extracellular vesicle release and promotes tumour metastasis during oxidative stress.

Reactive oxygen species (ROS)-induced oxidative DNA damages have been considered the main cause of mutations in genes, which are highly related to carcinogenesis and tumour progression. Extracellular vesicles play an important role in cancer metastasis. However, the precise role of DNA oxidative damage in extracellular vesicles (EVs)-mediated cancer cell migration and invasion remains unclear. Here, we reveal that ROS-mediated DNA oxidative damage signalling promotes tumour metastasis through increasing EVs release. Mechanistically, 8-oxoguanine DNA glycosylase (OGG1) recognises and binds to its substrate 8-oxo-7,8-dihydroguanine (8-oxoG), recruiting NF-κB to the synaptotagmin 7 (SYT7) promoter and thereby triggering SYT7 transcription. The upregulation of SYT7 expression leads to increased release of E-cadherin-loaded EVs, which depletes intracellular E-cadherin, thereby inducing epithelial-mesenchymal transition (EMT). Notably, Th5487, the inhibitor of DNA binding activity of OGG1, blocks the recognition and transmission of oxidative signals, alleviates SYT7 expression and suppresses EVs release, thereby preventing tumour progression in vitro and in vivo. Collectively, our study illuminates the significance of 8-oxoG/OGG1/SYT7 axis-driven EVs release in oxidative stress-induced tumour metastasis. These findings provide a deeper understanding of the molecular basis of cancer progression and offer potential avenues for therapeutic intervention.

Poly(G)7 box: a functional element of mammalian 18S rRNA involved in translation.

In eukaryotes, the ribosomal small subunit (40S) is composed of 18S rRNA and 33 ribosomal proteins. 18S rRNA has a special secondary structure and is an indispensable part of the translation process. Herein, a special sequence located in mammalian 18S rRNA named Poly(G)7box, which is composed of seven guanines, was found. Poly(G)7 can form a special and stable secondary structure by binding to the translation elongation factor subunit eEF1D and the ribosomal protein RPL32. Poly(G)7box was transfected into cells, and the translation efficiency of cells was inhibited. We believe that Poly(G)7box is an important translation-related functional element located on mammalian 18S rRNA, meanwhile the Poly(G)7 located on mRNA 5' and 3' box does not affect mRNA translation.

DNA mismatch repair controls the mutagenicity of Polymerase ζ-dependent translesion synthesis at methylated guanines.

By replicating damaged nucleotides, error-prone DNA translesion synthesis (TLS) enables the completion of replication, albeit at the expense of fidelity. TLS of helix-distorting DNA lesions, that usually have reduced capacity of basepairing, comprises insertion opposite the lesion followed by extension, the latter in particular by polymerase ζ (Pol ζ). However, little is known about involvement of Pol ζ in TLS of non- or poorly-distorting, but miscoding, lesions such as O6-methyldeoxyguanosine (O6-medG). Using purified Pol ζ we describe that the enzyme can misincorporate thymidine opposite O6-medG and efficiently extend from terminal mismatches, suggesting its involvement in the mutagenicity of O6-medG. Surprisingly, O6-medG lesions induced by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) appeared more, rather than less, mutagenic in Pol ζ-deficient mouse embryonic fibroblasts (MEFs) than in wild type MEFs. This suggested that in vivo Pol ζ participates in non-mutagenic TLS of O6-medG. However, we found that the Pol ζ-dependent misinsertions at O6-medG lesions are efficiently corrected by DNA mismatch repair (MMR), which masks the error-proneness of Pol ζ. We also found that the MNNG-induced mutational signature is determined by the adduct spectrum, and modulated by MMR. The signature mimicked single base substitution signature 11 in the catalogue of somatic mutations in cancer, associated with treatment with the methylating drug temozolomide. Our results unravel the individual roles of the major contributors to methylating drug-induced mutagenesis. Moreover, these results warrant caution as to the classification of TLS as mutagenic or error-free based on in vitro data or on the analysis of mutations induced in MMR-proficient cells.

HMGB3 and SUB1 Bind to and Facilitate the Repair of N2-Alkylguanine Lesions in DNA.

N2-Alkyl-2'-deoxyguanosine (N2-alkyl-dG) is a major type of minor-groove DNA lesions arising from endogenous metabolic processes and exogenous exposure to environmental contaminants. The N2-alkyl-dG lesions, if left unrepaired, can block DNA replication and transcription and induce mutations in these processes. Nevertheless, the repair pathways for N2-alkyl-dG lesions remain incompletely elucidated. By utilizing a photo-cross-linking coupled with mass spectrometry-based quantitative proteomic analysis, we identified a series of candidate N2-alkyl-dG-binding proteins. We found that two of these proteins, i.e., high-mobility group protein B3 (HMGB3) and SUB1, could bind directly to N2-nBu-dG-containing duplex DNA in vitro and promote the repair of this lesion in cultured human cells. In addition, HMGB3 and SUB1 protected cells against benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE). SUB1 exhibits preferential binding to both the cis and trans diastereomers of N2-BPDE-dG over unmodified dG. On the other hand, HMGB3 binds favorably to trans-N2-BPDE-dG; the protein, however, does not distinguish cis-N2-BPDE-dG from unmodified dG. Consistently, genetic ablation of HMGB3 conferred diminished repair of trans-N2-BPDE-dG, but not its cis counterpart, whereas loss of SUB1 conferred attenuated repair of both diastereomers. Together, we identified proteins involved in the cellular sensing and repair of minor-groove N2-alkyl-dG lesions and documented a unique role of HMGB3 in the stereospecific recognition and repair of N2-BPDE-dG.

What tunes guanine ionization potential in a nucleosome? An all-in-one systematic QM/MM assessment.

Guanine radical cations are precursors to oxidatively induced DNA lesions, and the determination of oxidative DNA hot spots beyond oligonucleotides remains a current challenge. In order to rationalize the finetuned ionization properties of the ∼60 guanines in a nucleosome core particle, we report a robust molecular dynamics-then-FO-DFTB/MM (fragment-orbital tight-binding density functional theory/molecular mechanics) simulation protocol spanning 20 µs. Our work allows us to identify several factors governing guanine ionization potential and map oxidative hotspots. Our results highlight the predominant role of the proximity of positively charged histone residues in the modulation of the guanine ionization potential up to 0.6 eV. Consequently, fast, long-range hole transfer in nucleosomal DNA could be tuned by the proximity of histone tails, which differs, from a biological point of view, on the chromatin state.

Unexpected large charge transfer rate mediated by adenine in twisted DNA structure.

DNA exhibits remarkable charge transfer ability, which is crucial for its biological functions and potential electronic applications. The charge transfer process in DNA is widely recognized as primarily mediated by guanine, while the contribution of other nucleobases is negligible. Using the tight-binding models in conjunction with first-principles calculations, we investigated the charge transfer behavior of homogeneous GC and AT pairs. We found that the charge transfer rate of adenine significantly changes. With overstretching, the charge transfer rate of adenine can even surpass that of guanine, by as much as five orders of magnitude at a twist angle of around 26°. Further analysis reveals that it is attributed to the turnover of the relative coupling strength between homogeneous GC and AT base pairs, which is caused by the symmetry exchange between the two highest occupied molecular orbitals of base pairs occurring at different twist angles. Given the high degree of flexibility of DNA in vivo and in vitro conditions, these findings prompt us to reconsider the mechanism of biological functions concerning the charge transfer in DNA molecules and further open the potential of DNA as a biomaterial for electronic applications.

Inhibition of Uracil DNA Glycosylase Alters Frequency and Spectrum of Action-at-a-Distance Mutations Induced by 8-Oxo-7,8-dihydroguanine.

The generation of DNA damage causes mutations and consequently cancer. Reactive oxygen species are important sources of DNA damage and some mutation signatures found in human cancers. 8-Oxo-7,8-dihydroguanine (GO, 8-hydroxyguanine) is one of the most abundant oxidized bases and induces a G→T transversion mutation at the modified site. The damaged G base also causes untargeted base substitution mutations at the G bases of 5'-GpA-3' dinucleotides (action-at-a-distance mutations) in human cells, and the cytosine deaminase apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3 (APOBEC3) is involved in the mutation process. The deaminated cytosine, i.e., uracil, bases are expected to be removed by uracil DNA glycosylase. Most of the substitution mutations at the G bases of 5'-GpA-3' might be caused by abasic sites formed by the glycosylase. In this study, we expressed the uracil DNA glycosylase inhibitor from Bacillus subtilis bacteriophage PBS2 in human U2OS cells and examined the effects on the GO-induced action-at-a-distance mutations. The inhibition of uracil DNA glycosylase increased the mutation frequency, and in particular, the frequency of G→A transitions. These results indicated that uracil DNA glycosylase, in addition to APOBEC3, is involved in the untargeted mutation process induced by GO.

FANCD2 counteracts O6-methylguanine-induced mismatch repair-dependent apoptosis.

BACKGROUND: Sn1-type alkylating agents methylate the oxygen atom on guanine bases thereby producing O6-methylguanine. This modified base could pair with thymine and cytosine, resulting in the formation of O6-methylguanine/thymine mismatch during DNA replication, recognized by the mismatch repair (MMR) complex, which then initiates the DNA damage response and subsequent apoptotic processes. In our investigation of the molecular mechanisms underlying MMR-dependent apoptosis, we observed FANCD2 modification upon the activity of alkylating agent N-methyl-N-nitrosourea (MNU). This observation led us to hypothesize a relevant role for FANCD2 in the apoptosis induction process. METHODS AND RESULTS: We generated FANCD2 knockout cells using the CRISPR/Cas9 method in the human cervical cancer cell line HeLa MR. FANCD2-deficient cells exhibited MNU hypersensitivity. Upon MNU exposure, FANCD2 colocalized with the MMR complex. MNU-treated FANCD2 knockout cells displayed severe S phase delay followed by increased G2/M arrest and MMR-dependent apoptotic cell death. Moreover, FANCD2 knockout cells exhibited impaired CtIP and RAD51 recruitment to the damaged chromatin and DNA double-strand break accumulation, indicated by simultaneously observed increased γH2AX signal and 53BP1 foci. CONCLUSIONS: Our data suggest that FANCD2 is crucial for recruiting homologous recombination factors to the sites of the MMR-dependent replication stress to resolve the arrested replication fork and counteract O6-methylguanine-triggered MMR-dependent apoptosis.

Accurate identification of 8-oxoguanine in RNA with single-nucleotide resolution using ligase-dependent qPCR.

8-oxoguanine (o8G), a prevalent oxidative modification in RNA induced by reactive oxygen species (ROS), plays a pivotal role in regulating RNA functions. Accurate detection and quantification of o8G modifications is critical to understanding their biological significance and potential as disease biomarkers, but effective detection methods remain limited. Here, we have developed a highly specific T3 DNA ligase-dependent qPCR assay that exploits the enzyme's ability to discriminate o8G from guanine (G) with single-nucleotide resolution. This method can detect o8G in RNA at levels as low as 500 fM, with an up to 18-fold higher selectivity for discriminating o8G from G. By simulating oxidative stress conditions in SH-SY5Y and HS683 cell lines treated with rotenone, we successfully identified site-specific o8G modifications in key miRNAs associated with neuroprotective responses, including miR-124, let-7a and miR-29a. The developed assay holds significant promise for the practical identification of o8G, facilitating its potential for detailed studies of o8G dynamics in various biological contexts and diseases.

Development of deaminase-free T-to-S base editor and C-to-G base editor by engineered human uracil DNA glycosylase.

DNA base editors enable direct editing of adenine (A), cytosine (C), or guanine (G), but there is no base editor for direct thymine (T) editing currently. Here we develop two deaminase-free glycosylase-based base editors for direct T editing (gTBE) and C editing (gCBE) by fusing Cas9 nickase (nCas9) with engineered human uracil DNA glycosylase (UNG) variants. By several rounds of structure-informed rational mutagenesis on UNG in cultured human cells, we obtain gTBE and gCBE with high activity of T-to-S (i.e., T-to-C or T-to-G) and C-to-G conversions, respectively. Furthermore, we conduct parallel comparison of gTBE/gCBE with those recently developed using other protein engineering strategies, and find gTBE/gCBE show the outperformance. Thus, we provide several base editors, gTBEs and gCBEs, with corresponding engineered UNG variants, broadening the targeting scope of base editors.

May DNA analyses be biased by hidden oxidative damage? Voltammetric study of temperature and oxidation stress effect.

The analysis of nucleic acids is one of the fundamental parts of modern molecular biology and molecular diagnostics. The information collected predominantly depends on the condition of the genetic material. All potential damage induced by oxidative stress may affect the final results of the analysis of genetic material obtained using commonly used techniques such as polymerase chain reaction or sequencing. The aim of this work was to evaluate the effects of high temperature and pH on DNA structure in the context of the occurrence of oxidative damage, using square-wave voltammetry and two independent research protocols. We resulted in visible oxidation damage registered in acidic conditions after the thermal denaturation process (pH 4.7) with changes in the intensity of guanine and adenine signals. However, using phosphate buffer (pH 7.0) for DNA denaturation negatively affected the DNA structure, but without any oxidized derivatives present. This leads to the conclusion that oxidation occurring in the DNA melting process results in the formation of various derivatives of nucleobases, both electrochemically active and inactive. These derivatives may distort the results of molecular tests due to the possibility of forming complementary bonds with various nucleobases. For example, 8-oxoguanine can form pairs with both cytosine and adenine.

Developing guanine base editors for G-to-T editing in rice.

Two guanine base editors created using an engineered N-methylpurine DNA glycosylase with CRISPR systems achieved targeted G-to-T editing with 4.94-12.50% efficiency in rice (Oryza sativa). The combined use of the DNA glycosylase and deaminases enabled co-editing of target guanines with adenines or cytosines.

Human CST Stimulates Base Excision Repair to Prevent the Accumulation of Oxidative DNA Damage.

CTC1-STN1-TEN1 (CST) is a single-stranded DNA binding protein vital for telomere length maintenance with additional genome-wide roles in DNA replication and repair. While CST was previously shown to function in double-strand break repair and promote replication restart, it is currently unclear whether it has specialized roles in other DNA repair pathways. Proper and efficient repair of DNA is critical to protecting genome integrity. Telomeres and other G-rich regions are strongly predisposed to oxidative DNA damage in the form of 8-oxoguanines, which are typically repaired by the base-excision repair (BER) pathway. Moreover, recent studies suggest that CST functions in the repair of oxidative DNA lesions. Therefore, we tested whether CST interacts with and regulates BER protein activity. Here, we show that CST robustly stimulates proteins involved in BER, including OGG1, Pol ß, APE1, and LIGI, on both telomeric and non-telomeric DNA substrates. Biochemical reconstitution of the pathway indicates that CST stimulates BER. Finally, knockout of STN1 or CTC1 leads to increased levels of 8-oxoguanine, suggesting defective BER in the absence of CST. Combined, our results define an undiscovered function of CST in BER, where it acts as a stimulatory factor to promote efficient genome-wide oxidative repair.

ArcS from Thermococcus kodakarensis transfers L-lysine to preQ0 nucleoside derivatives as minimum substrate RNAs.

Archaeosine (G+) is an archaea-specific tRNA modification synthesized via multiple steps. In the first step, archaeosine tRNA guanine transglucosylase (ArcTGT) exchanges the G15 base in tRNA with 7-cyano-7-deazaguanine (preQ0). In Euryarchaea, preQ015 in tRNA is further modified by archaeosine synthase (ArcS). Thermococcus kodakarensis ArcS catalyzes a lysine-transfer reaction to produce preQ0-lysine (preQ0-Lys) as an intermediate. The resulting preQ0-Lys15 in tRNA is converted to G+15 by a radical S-adenosyl-L-methionine enzyme for archaeosine formation (RaSEA), which forms a complex with ArcS. Here, we focus on the substrate tRNA recognition mechanism of ArcS. Kinetic parameters of ArcS for lysine and tRNA-preQ0 were determined using a purified enzyme. RNA fragments containing preQ0 were prepared from Saccharomyces cerevisiae tRNAPhe-preQ015. ArcS transferred 14C-labeled lysine to RNA fragments. Furthermore, ArcS transferred lysine to preQ0 nucleoside and preQ0 nucleoside 5'-monophosphate. Thus, the L-shaped structure and the sequence of tRNA are not essential for the lysine-transfer reaction by ArcS. However, the presence of D-arm structure accelerates the lysine-transfer reaction. Because ArcTGT from thermophilic archaea recognizes the common D-arm structure, we expected the combination of T. kodakarensis ArcTGT and ArcS and RaSEA complex would result in the formation of preQ0-Lys15 in all tRNAs. This hypothesis was confirmed using 46 T. kodakarensis tRNA transcripts and three Haloferax volcanii tRNA transcripts. In addition, ArcTGT did not exchange the preQ0-Lys15 in tRNA with guanine or preQ0 base, showing that formation of tRNA-preQ0-Lys by ArcS plays a role in preventing the reverse reaction in G+ biosynthesis.

o8G Site-Specifically Modified tRF-1-AspGTC: A Novel Therapeutic Target and Biomarker for Pulmonary Hypertension.

BACKGROUND: Hypoxia and oxidative stress contribute to the development of pulmonary hypertension (PH). tRNA-derived fragments play important roles in RNA interference and cell proliferation, but their epitranscriptional roles in PH development have not been investigated. We aimed to gain insight into the mechanistic contribution of oxidative stress-induced 8-oxoguanine in pulmonary vascular remodeling. METHODS: Through small RNA modification array analysis and quantitative polymerase chain reaction, a significant upregulation of the 8-oxoguanine -modified tRF-1-AspGTC was found in the lung tissues and the serum of patients with PH. RESULTS: This modification occurs at the position 5 of the tRF-1-AspGTC (5o8G tRF). Inhibition of the 5o8G tRF reversed hypoxia-induced proliferation and apoptosis resistance in pulmonary artery smooth muscle cells. Further investigation unveiled that the 5o8G tRF retargeted mRNA of WNT5A (Wingless-type MMTV integration site family, member 5A) and CASP3 (Caspase3) and inhibited their expression. Ultimately, BMPR2 (Bone morphogenetic protein receptor 2) -reactive oxygen species/5o8G tRF/WNT5A signaling pathway exacerbated the progression of PH. CONCLUSIONS: Our study highlights the role of site-specific 8-oxoguanine-modified tRF in promoting the development of PH. Our findings present a promising therapeutic avenue for managing PH and propose 5o8G tRF as a potential innovative marker for diagnosing this disease.

Metagenome mining and functional analysis reveal oxidized guanine DNA repair at the Lost City Hydrothermal Field.

The GO DNA repair system protects against GC → TA mutations by finding and removing oxidized guanine. The system is mechanistically well understood but its origins are unknown. We searched metagenomes and abundantly found the genes encoding GO DNA repair at the Lost City Hydrothermal Field (LCHF). We recombinantly expressed the final enzyme in the system to show MutY homologs function to suppress mutations. Microbes at the LCHF thrive without sunlight, fueled by the products of geochemical transformations of seafloor rocks, under conditions believed to resemble a young Earth. High levels of the reductant H2 and low levels of O2 in this environment raise the question, why are resident microbes equipped to repair damage caused by oxidative stress? MutY genes could be assigned to metagenome-assembled genomes (MAGs), and thereby associate GO DNA repair with metabolic pathways that generate reactive oxygen, nitrogen and sulfur species. Our results indicate that cell-based life was under evolutionary pressure to cope with oxidized guanine well before O2 levels rose following the great oxidation event.

The role of DNA and RNA guanosine oxidation in cardiovascular diseases.

Cardiovascular diseases (CVD) persist as a prominent cause of mortality worldwide, with oxidative stress constituting a pivotal contributory element. The oxidative modification of guanosine, specifically 8-oxoguanine, has emerged as a crucial biomarker for oxidative stress, providing novel insights into the molecular underpinnings of CVD. 8-Oxoguanine can be directly generated at the DNA (8-oxo-dG) and RNA (8-oxo-G) levels, as well as at the free nucleotide level (8-oxo-dGTP or 8-oxo-GTP), which are produced and can be integrated through DNA replication or RNA transcription. When exposed to oxidative stress, guanine is more readily produced in RNA than in DNA. A burgeoning body of research surrounds 8-oxoguanine, exhibits its accumulation playing a pivotal role in the development of CVD. Therapeutic approaches targeting oxidative 8-Oxoguanine damage to DNA and RNA, encompassing the modulation of repair enzymes and the development of small molecule inhibitors, are anticipated to enhance CVD management. In conclusion, we explore the noteworthy elevation of 8-oxoguanine levels in patients with various cardiac conditions and deliberate upon the formation and regulation of 8-oxo-dG and 8-oxo-G under oxidative stress, as well as their function in CVD.

A new reagent for in vivo structure probing of RNA G and U residues that improves RNA structure prediction alone and combined with DMS.

A key to understanding the roles of RNA in regulating gene expression is knowing their structures in vivo. One way to obtain this information is through probing the structures of RNA with chemicals. To probe RNA structure directly in cells, membrane-permeable reagents that modify the Watson-Crick (WC) face of unpaired nucleotides can be used. Although dimethyl sulfate (DMS) has led to substantial insight into RNA structure, it has limited nucleotide specificity in vivo, with WC face reactivity only at adenine (A) and cytosine (C) at neutral pH. The reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was recently shown to modify the WC face of guanine (G) and uracil (U). Although useful at lower concentrations in experiments that measure chemical modifications by reverse transcription (RT) stops, at higher concentrations necessary for detection by mutational profiling (MaP), EDC treatment leads to degradation of RNA. Here, we demonstrate EDC-stimulated degradation of RNA in Gram-negative and Gram-positive bacteria. In an attempt to overcome these limitations, we developed a new carbodiimide reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide (ETC), which we show specifically modifies unpaired Gs and Us in vivo without substantial degradation of RNA. We establish ETC as a probe for MaP and optimize the RT conditions and computational analysis in Escherichia coli Importantly, we demonstrate the utility of ETC as a probe for improving RNA structure prediction both alone and with DMS.

Integrated data from intravital imaging and HPLC-MS/MS analysis reveal large interspecies differences in AFB1 metabolism in mice and rats.

Large interspecies differences between rats and mice concerning the hepatotoxicity and carcinogenicity of aflatoxin B1 (AFB1) are known, with mice being more resistant. However, a comprehensive interspecies comparison including subcellular liver tissue compartments has not yet been performed. In this study, we performed spatio-temporal intravital analysis of AFB1 kinetics in the livers of anesthetized mice and rats. This was supported by time-dependent analysis of the parent compound as well as metabolites and adducts in blood, urine, and bile of both species by HPLC-MS/MS. The integrated data from intravital imaging and HPLC-MS/MS analysis revealed major interspecies differences between rats and mice: (1) AFB1-associated fluorescence persisted much longer in the nuclei of rat than mouse hepatocytes; (2) in the sinusoidal blood, AFB1-associated fluorescence was rapidly cleared in mice, while a time-dependent increase was observed in rats in the first three hours after injection followed by a plateau that lasted until the end of the observation period of six hours; (3) this coincided with a far stronger increase of AFB1-lysine adducts in the blood of rats compared to mice; (4) the AFB1-guanine adduct was detected at much higher concentrations in bile and urine of rats than mice. In both species, the AFB1-glutathione conjugate was efficiently excreted via bile, where it reached concentrations at least three orders of magnitude higher compared to blood. In conclusion, major differences between mice and rats were observed, concerning the nuclear persistence, formation of AFB1-lysine adducts, and the AFB1-guanine adducts.

Mapping the transporter-substrate interactions of the Trypanosoma cruzi NB1 nucleobase transporter reveals the basis for its high affinity and selectivity for hypoxanthine and guanine and lack of nucleoside uptake.

Trypanosoma cruzi is a protozoan parasite and the etiological agent of Chagas disease, a debilitating and sometimes fatal disease that continues to spread to new areas. Yet, Chagas disease is still only treated with two related nitro compounds that are insufficiently effective and cause severe side effects. Nucleotide metabolism is one of the known vulnerabilities of T. cruzi, as they are auxotrophic for purines, and nucleoside analogues have been shown to have genuine promise against this parasite in vitro and in vivo. Since purine antimetabolites require efficient uptake through transporters, we here report a detailed characterisation of the T. cruzi NB1 nucleobase transporter with the aim of elucidating the interactions between TcrNB1 and its substrates and finding the positions that can be altered in the design of novel antimetabolites without losing transportability. Systematically determining the inhibition constants (Ki) of purine analogues for TcrNB1 yielded their Gibbs free energy of interaction, ΔG0. Pairwise comparisons of substrate (hypoxanthine, guanine, adenine) and analogues allowed us to determine that optimal binding affinity by TcrNB1 requires interactions with all four nitrogen residues of the purine ring, with N1 and N9, in protonation state, functioning as presumed hydrogen bond donors and unprotonated N3 and N7 as hydrogen bond acceptors. This is the same interaction pattern as we previously described for the main nucleobase transporters of Trypanosoma brucei spp. and Leishmania major and makes it the first of the ENT-family genes that is functionally as well as genetically conserved between the three main kinetoplast pathogens.