The cytidine deaminase APOBEC3C has unique sequence and genome feature preferences.

APOBEC proteins are cytidine deaminases that restrict the replication of viruses and transposable elements. Several members of the APOBEC3 family, APOBEC3A, APOBEC3B, and APOBEC3H-I, can access the nucleus and cause what is thought to be indiscriminate deamination of the genome, resulting in mutagenesis and genome instability. Although APOBEC3C is also present in the nucleus, the full scope of its deamination target preferences is unknown. By expressing human APOBEC3C in a yeast model system, I have defined the APOBEC3C mutation signature, as well as the preferred genome features of APOBEC3C targets. The APOBEC3C mutation signature is distinct from those of the known cancer genome mutators APOBEC3A and APOBEC3B. APOBEC3C produces DNA strand-coordinated mutation clusters, and APOBEC3C mutations are enriched near the transcription start sites of active genes. Surprisingly, APOBEC3C lacks the bias for the lagging strand of DNA replication that is seen for APOBEC3A and APOBEC3B. The unique preferences of APOBEC3C constitute a mutation profile that will be useful in defining sites of APOBEC3C mutagenesis in human genomes.

RNA 2'-O-methylation promotes persistent R-loop formation and AID-mediated IgH class switch recombination.

BACKGROUND: RNA-DNA hybrids or R-loops are associated with deleterious genomic instability and protective immunoglobulin class switch recombination (CSR). However, the underlying phenomenon regulating the two contrasting functions of R-loops is unknown. Notably, the underlying mechanism that protects R-loops from classic RNase H-mediated digestion thereby promoting persistence of CSR-associated R-loops during CSR remains elusive. RESULTS: Here, we report that during CSR, R-loops formed at the immunoglobulin heavy (IgH) chain are modified by ribose 2'-O-methylation (2'-OMe). Moreover, we find that 2'-O-methyltransferase fibrillarin (FBL) interacts with activation-induced cytidine deaminase (AID) associated snoRNA aSNORD1C to facilitate the 2'-OMe. Moreover, deleting AID C-terminal tail impairs its association with aSNORD1C and FBL. Disrupting FBL, AID or aSNORD1C expression severely impairs 2'-OMe, R-loop stability and CSR. Surprisingly, FBL, AID's interaction partner and aSNORD1C promoted AID targeting to the IgH locus. CONCLUSION: Taken together, our results suggest that 2'-OMe stabilizes IgH-associated R-loops to enable productive CSR. These results would shed light on AID-mediated CSR and explain the mechanism of R-loop-associated genomic instability.

The SMC5/6 complex prevents genotoxicity upon APOBEC3A-mediated replication stress.

Mutational patterns caused by APOBEC3 cytidine deaminase activity are evident throughout human cancer genomes. In particular, the APOBEC3A family member is a potent genotoxin that causes substantial DNA damage in experimental systems and human tumors. However, the mechanisms that ensure genome stability in cells with active APOBEC3A are unknown. Through an unbiased genome-wide screen, we define the Structural Maintenance of Chromosomes 5/6 (SMC5/6) complex as essential for cell viability when APOBEC3A is active. We observe an absence of APOBEC3A mutagenesis in human tumors with SMC5/6 dysfunction, consistent with synthetic lethality. Cancer cells depleted of SMC5/6 incur substantial genome damage from APOBEC3A activity during DNA replication. Further, APOBEC3A activity results in replication tract lengthening which is dependent on PrimPol, consistent with re-initiation of DNA synthesis downstream of APOBEC3A-induced lesions. Loss of SMC5/6 abrogates elongated replication tracts and increases DNA breaks upon APOBEC3A activity. Our findings indicate that replication fork lengthening reflects a DNA damage response to APOBEC3A activity that promotes genome stability in an SMC5/6-dependent manner. Therefore, SMC5/6 presents a potential therapeutic vulnerability in tumors with active APOBEC3A.

A real-time biochemical assay for quantitative analyses of APOBEC-catalyzed DNA deamination.

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics. The assay also offers a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

Conquering new grounds: plant organellar C-to-U RNA editing factors can be functional in the plant cytosol.

Plant mitochondrial and chloroplast transcripts are subject to numerous events of specific cytidine-to-uridine (C-to-U) RNA editing to correct genetic information. Key protein factors for this process are specific RNA-binding pentatricopeptide repeat (PPR) proteins, which are encoded in the nucleus and post-translationally imported into the two endosymbiotic organelles. Despite hundreds of C-to-U editing sites in the plant organelles, no comparable editing has been found for nucleo-cytosolic mRNAs raising the question why plant RNA editing is restricted to chloroplasts and mitochondria. Here, we addressed this issue in the model moss Physcomitrium patens, where all PPR-type RNA editing factors comprise specific RNA-binding and cytidine deamination functionalities in single proteins. To explore whether organelle-type RNA editing can principally also take place in the plant cytosol, we expressed PPR56, PPR65 and PPR78, three editing factors recently shown to also function in a bacterial setup, together with cytosolic co-transcribed native targets in Physcomitrium. While we obtained unsatisfying results upon their constitutive expression, we found strong cytosolic RNA editing under hormone-inducible expression. Moreover, RNA-Seq analyses revealed varying numbers of up to more than 900 off-targets in other cytosolic transcripts. We conclude that PPR-mediated C-to-U RNA editing is not per se incompatible with the plant cytosol but that its limited target specificity has restricted its occurrence to the much less complex transcriptomes of mitochondria and chloroplast in the course of evolution.

AID in non-Hodgkin B-cell lymphomas: The consequences of on- and off-target activity.

Activation induced cytidine deaminase (AID) is a key element of the adaptive immune system, required for immunoglobulin isotype switching and affinity maturation of B-cells as they undergo the germinal center (GC) reaction in peripheral lymphoid tissue. The inherent DNA damaging activity of this enzyme can also have off-target effects in B-cells, producing lymphomagenic chromosomal translocations that are characteristic features of various classes of non-Hodgkin B-cell lymphoma (B-NHL), and generating oncogenic mutations, so-called aberrant somatic hypermutation (aSHM). Additionally, AID has been found to affect gene expression through demethylation as well as altered interactions between gene regulatory elements. These changes have been most thoroughly studied in B-NHL arising from GC B-cells. Here, we describe the most common classes of GC-derived B-NHL and explore the consequences of on- and off-target AID activity in B and plasma cell neoplasms. The relationships between AID expression, including effects of infection and other exposures/agents, mutagenic activity and lymphoma biology are also discussed.

A real-time biochemical assay for quantitative analyses of APOBEC-catalyzed DNA deamination.

Over the past decade, the connection between APOBEC3 cytosine deaminases and cancer mutagenesis has become increasingly apparent. This growing awareness has created a need for biochemical tools that can be used to identify and characterize potential inhibitors of this enzyme family. In response to this challenge, we have developed a Real-time APOBEC3-mediated DNA Deamination (RADD) assay. This assay offers a single-step set-up and real-time fluorescent read-out, and it is capable of providing insights into enzyme kinetics and also offering a high-sensitivity and easily scalable method for identifying APOBEC3 inhibitors. This assay serves as a crucial addition to the existing APOBEC3 biochemical and cellular toolkit and possesses the versatility to be readily adapted into a high-throughput format for inhibitor discovery.

The Disassociation of A3G-Related HIV-1 cDNA G-to-A Hypermutation to Viral Infectivity.

APOBEC3G (A3G) restricts HIV-1 replication primarily by reducing viral cDNA and inducing G-to-A hypermutations in viral cDNA. HIV-1 encodes virion infectivity factor (Vif) to counteract A3G primarily by excluding A3G viral encapsidation. Even though the Vif-induced exclusion is robust, studies suggest that A3G is still detectable in the virion. The impact of encapsidated A3G in the HIV-1 replication is unclear. Using a highly sensitive next-generation sequencing (NGS)-based G-to-A hypermutation detecting assay, we found that wild-type HIV-1 produced from A3G-expressing T-cells induced higher G-to-A hypermutation frequency in viral cDNA than HIV-1 from non-A3G-expressing T-cells. Interestingly, although the virus produced from A3G-expressing T-cells induced higher hypermutation frequency, there was no significant difference in viral infectivity, revealing a disassociation of cDNA G-to-A hypermutation to viral infectivity. We also measured G-to-A hypermutation in the viral RNA genome. Surprisingly, our data showed that hypermutation frequency in the viral RNA genome was significantly lower than in the integrated DNA, suggesting a mechanism exists to preferentially select intact genomic RNA for viral packing. This study revealed a new insight into the mechanism of HIV-1 counteracting A3G antiviral function and might lay a foundation for new antiviral strategies.

Loss of cytidine deaminase expression as a potential attempt to counteract the process of carcinogenesis by reducing basal PARP-1 activity and increasing tau levels.

Cytidine deaminase (CDA) is a pyrimidine salvage pathway enzyme that catalyzes the hydrolytic deamination of free cytidine and deoxycytidine to uridine and deoxyuridine, respectively. Our team discovered that CDA deficiency is associated with several aspects of genetic instability, such as increased sister chromatid exchange and ultrafine anaphase bridge frequencies. Based on these results, we sought (1) to determine how CDA deficiency contributes to genetic instability, (2) to explore the possible relationships between CDA deficiency and carcinogenesis, and (3) to develop a new anticancer treatment targeting CDA-deficient tumors. This review summarizes our major findings indicating that CDA deficiency is associated with a genetic instability that does not confer an increased cancer risk. In light of our results and published data, I propose a novel hypothesis that loss of CDA, by reducing basal PARP-1 activity and increasing Tau levels, may reflect an attempt to prevent, slow or reverse the process of carcinogenesis.

Synthesis of 1,4-azaphosphinine nucleosides and evaluation as inhibitors of human cytidine deaminase and APOBEC3A.

Nucleoside and polynucleotide cytidine deaminases (CDAs), such as CDA and APOBEC3, share a similar mechanism of cytosine to uracil conversion. In 1984, phosphapyrimidine riboside was characterised as the most potent inhibitor of human CDA, but the quick degradation in water limited the applicability as a potential therapeutic. To improve stability in water, we synthesised derivatives of phosphapyrimidine nucleoside having a CH2 group instead of the N3 atom in the nucleobase. A charge-neutral phosphinamide and a negatively charged phosphinic acid derivative had excellent stability in water at pH 7.4, but only the charge-neutral compound inhibited human CDA, similar to previously described 2'-deoxyzebularine (Ki = 8.0 ± 1.9 and 10.7 ± 0.5 µM, respectively). However, under basic conditions, the charge-neutral phosphinamide was unstable, which prevented the incorporation into DNA using conventional DNA chemistry. In contrast, the negatively charged phosphinic acid derivative was incorporated into DNA instead of the target 2'-deoxycytidine using an automated DNA synthesiser, but no inhibition of APOBEC3A was observed for modified DNAs. Although this shows that the negative charge is poorly accommodated in the active site of CDA and APOBEC3, the synthetic route reported here provides opportunities for the synthesis of other derivatives of phosphapyrimidine riboside for potential development of more potent CDA and APOBEC3 inhibitors.

Secreted novel AID/APOBEC-like deaminase 1 (SNAD1) - a new important player in fish immunology.

The AID/APOBECs are a group of zinc-dependent cytidine deaminases that catalyse the deamination of bases in nucleic acids, resulting in a cytidine to uridine transition. Secreted novel AID/APOBEC-like deaminases (SNADs), characterized by the presence of a signal peptide are unique among all of intracellular classical AID/APOBECs, which are the central part of antibody diversity and antiviral defense. To date, there is no available knowledge on SNADs including protein characterization, biochemical characteristics and catalytic activity. We used various in silico approaches to define the phylogeny of SNADs, their common structural features, and their potential structural variations in fish species. Our analysis provides strong evidence of the universal presence of SNAD1 proteins/transcripts in fish, in which expression commences after hatching and is highest in anatomical organs linked to the immune system. Moreover, we searched published fish data and identified previously, "uncharacterized proteins" and transcripts as SNAD1 sequences. Our review into immunological research suggests SNAD1 role in immune response to infection or immunization, and interactions with the intestinal microbiota. We also noted SNAD1 association with temperature acclimation, environmental pollution and sex-based expression differences, with females showing higher level. To validate in silico predictions we performed expression studies of several SNAD1 gene variants in carp, which revealed distinct patterns of responses under different conditions. Dual sensitivity to environmental and pathogenic stress highlights its importance in the fish and potentially enhancing thermotolerance and immune defense. Revealing the biological roles of SNADs represents an exciting new area of research related to the role of DNA and/or RNA editing in fish biology.

Combining a Base Deaminase Mutator with Phage-Assisted Evolution.

Phage-assisted evolution has emerged as a powerful technique for improving a protein's function by using mutagenesis and selective pressure. However, mutations typically occur throughout the host's genome and are not limited to the gene-of-interest (GOI): these undesirable genomic mutations can yield host cells that circumvent the system's selective pressure. Our system targets mutations specifically toward the GOI by combining T7 targeted mutagenesis and phage-assisted evolution. This system improves the structure and function of proteins by accumulating favorable mutations that can change its binding affinity, specificity, and activity.

Deciphering the role of Enterococcus faecium cytidine deaminase in gemcitabine resistance of gallbladder cancer.

Gemcitabine-based chemotherapy is a cornerstone of standard care for gallbladder cancer (GBC) treatment. Still, drug resistance remains a significant challenge, influenced by factors such as tumor-associated microbiota impacting drug concentrations within tumors. Enterococcus faecium, a member of tumor-associated microbiota, was notably enriched in the GBC patient cluster. In this study, we investigated the biochemical characteristics, catalytic activity, and kinetics of the cytidine deaminase of E. faecium (EfCDA). EfCDA showed the ability to convert gemcitabine to its metabolite 2',2'-difluorodeoxyuridine. Both EfCDA and E. faecium can induce gemcitabine resistance in GBC cells. Moreover, we determined the crystal structure of EfCDA, in its apo form and in complex with 2', 2'-difluorodeoxyuridine at high resolution. Mutation of key residues abolished the catalytic activity of EfCDA and reduced the gemcitabine resistance in GBC cells. Our findings provide structural insights into the molecular basis for recognizing gemcitabine metabolite by a bacteria CDA protein and may provide potential strategies to combat cancer drug resistance and improve the efficacy of gemcitabine-based chemotherapy in GBC treatment.

DNA flexibility can shape the preferential hypermutation of antibody genes.

Antibody-coding genes accumulate somatic mutations to achieve antibody affinity maturation. Genetic dissection using various mouse models has shown that intrinsic hypermutations occur preferentially and are predisposed in the DNA region encoding antigen-contacting residues. The molecular basis of nonrandom/preferential mutations is a long-sought question in the field. Here, we summarize recent findings on how single-strand (ss)DNA flexibility facilitates activation-induced cytidine deaminase (AID) activity and fine-tunes the mutation rates at a mesoscale within the antibody variable domain exon. We propose that antibody coding sequences are selected based on mutability during the evolution of adaptive immunity and that DNA mechanics play a noncoding role in the genome. The mechanics code may also determine other cellular DNA metabolism processes, which awaits future investigation.

Genomic Characteristics and Its Therapeutic Implications in Breast Cancer Patients with Detectable Molecular Residual Disease.

PURPOSE: Molecular residual disease (MRD) is the main cause of postoperative recurrence of breast cancer. However, the baseline tumor genomic characteristics and therapeutic implications of breast cancer patients with detectable MRD after surgery are still unknown. MATERIALS AND METHODS: In this study, we enrolled 80 patients with breast cancer who underwent next-generation sequencing-based genetic testing of 1,021 cancer-related genes performed on baseline tumor and postoperative plasma, among which 18 patients had detectable MRD after surgery. RESULTS: Baseline clinical characteristics found that patients with higher clinical stages were more likely to have detectable MRD. Analysis of single nucleotide variations and small insertions/deletions in baseline tumors showed that somatic mutations in MAP3K1, ATM, FLT1, GNAS, POLD1, SPEN, and WWP2 were significantly enriched in patients with detectable MRD. Oncogenic signaling pathway analysis revealed that alteration of the Cell cycle pathway was more likely to occur in patients with detectable MRD (p=0.012). Mutational signature analysis showed that defective DNA mismatch repair and activation-induced cytidine deaminase (AID) mediated somatic hypermutation (SHM) were associated with detectable MRD. According to the OncoKB database, 77.8% (14/18) of patients with detectable MRD had U.S. Food and Drug Administration-approved mutational biomarkers and targeted therapy. CONCLUSION: Our study reports genomic characteristics of breast cancer patients with detectable MRD. The cell cycle pathway, defective DNA mismatch repair, and AID-mediated SHM were found to be the possible causes of detectable MRD. We also found the vast majority of patients with detectable MRD have the opportunity to access targeted therapy.

Association of Cytidine Deaminase Polymorphism with Capecitabine Effectiveness in Breast Cancer Patients.

OBJECTIVE: Cancer is caused by abnormal growth and disruption of homeostatic mechanisms. Breast cancer is a major health problem and the second leading cause of death in women. Capecitabine is a prodrug of 5-fluorouracil, which is a non-cytotoxic agent and is used to treat advanced or metastatic breast cancer. Cytidine deaminase (CDA) plays an important role in the activation of capecitabine by acetylating 5-deoxycytidine in the liver, which ultimately leads to the formation of 5-fluorouracil. In this study, we aimed to investigate the relationship between cytidine deaminase polymorphism and capecitabine efficacy in breast cancer patients. METHODS: One hundred breast cancer patients aged 45 to 75 years were included in this study, all of whom received capecitabine as monotherapy. The serum levels of CA15.3, calcium, and estradiol E2 in breast cancer patients were investigated. In addition, the relationship of these markers with cytidine deaminase polymorphism was investigated in order to show the association of cytidine deaminase polymorphism with capecitabine efficacy in breast cancer patients. RESULT: The findings of this study showed that there is a statistically significant relationship between CDA enzyme polymorphisms (rs2072671 and rs532545) with estradiol and CA15.3 serum levels and a non-significant relationship with calcium level. Furthermore, patients who participated were highly polymorphic and heterozygotes genotypes had the highest frequency in both rs2072671 and rs532545 polymorphisms. CONCLUSION: The results obtained from the present study identified different genetic polymorphisms in the gene encoding the CDA enzyme. Overall, our results clearly showed direct evidence for the association of cytidine deaminase polymorphism with the efficacy of capecitabine in breast cancer patients, which could be useful in reducing side effects and improving drug response to capecitabine.

Engineered deaminases as a key component of DNA and RNA editing tools.

Over recent years, zinc-dependent deaminases have attracted increasing interest as key components of nucleic acid editing tools that can generate point mutations at specific sites in either DNA or RNA by combining a targeting module (such as a catalytically impaired CRISPR-Cas component) and an effector module (most often a deaminase). Deaminase-based molecular tools are already being utilized in a wide spectrum of therapeutic and research applications; however, their medical and biotechnological potential seems to be much greater. Recent reports indicate that the further development of nucleic acid editing systems depends largely on our ability to engineer the substrate specificity and catalytic activity of the editors themselves. In this review, we summarize the current trends and achievements in deaminase engineering. The presented data indicate that the potential of these enzymes has not yet been fully revealed or understood. Several examples show that even relatively minor changes in the structure of deaminases can give them completely new and unique properties.

Evaluation of potential role of R-loop and G-quadruplex DNA in the fragility of c-MYC during chromosomal translocation associated with Burkitt's lymphoma.

t(8;14) translocation is the hallmark of Burkitt's lymphoma and results in c-MYC deregulation. During the translocation, c-MYC gene on chromosome 8 gets juxtaposed to the Ig switch regions on chromosome 14. Although the promoter of c-MYC has been investigated for its mechanism of fragility, little is known about other c-MYC breakpoint regions. We have analyzed the translocation break points at the exon 1/intron 1 of c-MYC locus from patients with Burkitt's lymphoma. Results showed that the breakpoint region, when present on a plasmid, could fold into an R-loop confirmation in a transcription-dependent manner. Sodium bisulfite modification assay revealed significant single-strandedness on chromosomal DNA of Burkitt's lymphoma cell line, Raji, and normal lymphocytes, revealing distinct R-loops covering up to 100 bp region. Besides, ChIP-DRIP analysis reveals that the R-loop antibody can bind to the breakpoint region. Further, we show the formation of stable parallel intramolecular G-quadruplex on non-template strand of the genome. Finally, incubation of purified AID in vitro or overexpression of AID within the cells led to enhanced mutation frequency at the c-MYC breakpoint region. Interestingly, anti-γH2AX can bind to DSBs generated at the c-MYC breakpoint region within the cells. The formation of R-loop and G-quadruplex was found to be mutually exclusive. Therefore, our results suggest that AID can bind to the single-stranded region of the R-loop and G4 DNA, leading to the deamination of cytosines to uracil and induction of DNA breaks in one of the DNA strands, leading to double-strand break, which could culminate in t(8;14) chromosomal translocation.

Clinical use of biomarkers in the field of cytotoxic nucleoside analogues.

OBJECTIVES: Cytotoxic nucleosides (gemcitabine, cytarabine…) are used for the treatment of various malignancies. Their activity is dependent on the interaction with several proteins and enzymes of nucleotide metabolism. It has for a long time been hypothesized that the clinical activity of nucleoside analogues can be predicted by studying corresponding genes or gene products in clinical samples. METHODS: In this short review, I will present old and new published data from our group and others about the prediction of activity of these drugs concentrating on gene-candidate approaches, and discuss biological and technical limitations of these. RESULTS: A large number of studies have been conducted in various clinical settings (drugs, disease, patient cohort…) evaluating DNA, mRNA or protein-related markers. Although some individual parameters and associations thereof have been validated, only a very few numbers have been implemented in pretreatment evaluations of patients. CONCLUSION: There is still much to do in the field of outcome-prediction with nucleoside analogues. The use of multiparametric methods could increase the success rate but at the cost of a poorer understanding of molecular mechanisms.

Functional Assay for Measuring Bacterial Degradation of Gemcitabine Chemotherapy.

Drug biotransformation by the host microbiome can impact the therapeutic success of treatment. In the context of cancer, drug degradation can take place within the microenvironment of the targeted tumor by intratumor bacteria. In pancreatic cancer, increased chemo-resistance against the frontline chemotherapy gemcitabine is thought to arise from drug degradation by the tumor microbiome. This bacterial-drug interaction highlights the need for developing rapid assays for monitoring bacterial gemcitabine breakdown. While chemical approaches such as high-performance liquid chromatography are suitable for this task, they require specialized equipment and expertise and are limited in throughput. Functional cell-based assays represent an alternate approach for performing this task. We developed a functional assay to monitor the rate of bacterial gemcitabine breakdown using a highly sensitive bacterial reporter strain. Our method relies on standard laboratory equipment and can be implemented at high throughput to monitor drug breakdown by hundreds of strains simultaneously. This functional assay can be readily adapted to monitor degradation of other drugs. Key features Quantification of gemcitabine breakdown by incubating bacteria that degrades the drug and subsequently testing the growth of a reporter strain on filtered supernatant. Use of an optimized reporter strain that was genetically engineered to be a non-degrader strain and highly sensitive to gemcitabine. A high-throughput assay performed in microplates that can be adjusted for identifying bacteria with a fast or slow gemcitabine degradation rate. The assay results can be compared to results from a standard curve with known drug concentrations to quantify degradation rate.