Cytidine Deaminase Research Articles

BRCA2 plays a critical role in stabilizing stalled replication forks, yet critical gaps remain in understanding how BRCA2 deficiency triggers fork collapse and drives genomic instability. Here, we identify cytidine deaminase APOBEC3B as a key driver of this process. Using a unique uracil-in-DNA probe, we show that BRCA2 loss promotes APOBEC3B-mediated uracil accumulation in single-stranded DNA (U-ssDNA) at stalled forks. These lesions when processed by UNG2 and APE1, trigger fork collapse and release ssDNA fragments into the cytoplasm, activating NF-κB signaling. This in turn upregulates APOBEC3B expression, establishing a self-reinforcing loop that amplifies cytidine deamination at stalled forks and exacerbates genomic instability. Depletion of APOBEC3B, UNG2, or APE1 rescues these defects. Notably, BRCA1-deficient cells do not accumulate U-ssDNA or induce APOBEC3B under replication stress, highlighting a BRCA2-specific vulnerability. Clinically, low APE1 expression correlates with poor survival in patients with BRCA2-mutant tumors, with high APOBEC3 levels further worsening outcomes. Together, our findings establish that replication stress, whether intrinsic or therapy induced, triggers APOBEC3B overexpression and potentially activates an APOBEC3B-driven mutagenic loop in BRCA2-deficient cells. These results position APOBEC3B, UNG2 and APE1 as critical regulators of BRCA2-mutant tumor evolution and therapy resistance.

Cytidine analogues have conferred highly efficacious antimetabolites with broad utility as antiviral and anticancer agents. However, in many cases, human cytidine deaminase (CDA) converts the cytidine-based inhibitor into an inactive uridine metabolite with diminished potency. Inhibitors of CDA are useful agents to boost the efficacy of cytosine- and cytidine-containing drugs by inhibiting their rapid degradation. Toward the goal of developing CDA inhibitors, and our overarching interest in cytosine deaminase enzymes in general, we developed a real-time fluorescence-based deamination activity assay for CDA using isomorphic nucleoside analogues. Base-modified pyrimidine nucleosides that exhibit differential fluorescence properties as either the cytosine or uracil nucleobase were developed. We found that 5-benzo-2-furyl-2'-deoxycytidine is the best fluorescence reporter when implemented in a CDA enzyme activity assay, which permits detailed measurements of the kinetics of CDA activity in the presence or absence of inhibitors. Utilizing this assay, we then screened our in-house collection of 1054 fragments and found 23 hits that were further studied. Two fragment-sized CDA inhibitors with low micromolar potency (200-300 μM) and good ligand efficiency (>0.3) were identified, thereby conferring promising starting points for future inhibitor development.

Long Interspersed Nuclear Elements-1 (LINE-1 or L1) make up approximately 21% of the human genome, with some L1 loci containing intact open reading frames (ORFs) that facilitate retrotransposition. Because retrotransposition can have deleterious effects leading to mutations and genomic instability, L1 activity is typically suppressed in somatic cells through transcriptional and post-transcriptional mechanisms. However, L1 elements are derepressed in senescent cells causing age-associated inflammation. Despite the recognition of L1 activity as a hallmark of aging, the underlying molecular mechanisms governing L1 derepression in these cells are not fully understood. In this study, we employed high throughput sequencing datasets and validated our findings through independent experiments to investigate the regulation of L1 elements in senescent cells. Our results reveal that both replicative and oncogene-induced senescence are associated with reduced expression of the cytidine deaminase APOBEC3B, a known suppressor of L1 retrotransposition. Consequently, senescent cells exhibited diminished levels of C-to-U editing of full-length L1 elements. Moreover, Ribo-seq profiling indicated that progression to senescence is not only associated with increased L1 transcription, but also translation of L1 ORFs. In summary, our results suggest that the depletion of APOBEC3B contributes to enhanced activity of L1 in senescent cells and promotion of L1-induced DNA damage and aging.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal cancer with poor prognosis. COP9 signalosome subunit 6 (CSN6), a key regulator of different E3 ubiquitin ligases, plays oncogenic roles in various cancers. However, its function in PDAC remains elusive. Here, this demonstrates that human PDAC tumors expressing high levels of CSN6 present with poor prognosis and gemcitabine resistance. Conditional knockout (KO) of CSN6 hinders tumor formation in a KPP spontaneous PDAC mouse model. Proteomic analysis indicates that CSN6 promotes ribosome biogenesis by activating rDNA transcription and protein synthesis. Mechanistically, CSN6 antagonizes DDB1-CUL4 associated factor 1 (DCAF1)-mediated ubiquitination of Nucleophosmin (NPM1), thereby promoting NPM1-orchestrated ribosome biogenesis. In line with CSN6-mediated gemcitabine resistance, CSN6-NPM1 axis enhances ribosome biogenesis, thereby promoting translation of gemcitabine resistance genes, including Cytidine deaminase (CDA), Ribonucleotide reductase subunit M1/2 (RRM1/2). Significantly, combining gemcitabine with NPM1 inhibitor NSC348884 synergistically suppresses CSN6-high pancreatic cancer xenografts. Clinically, CSN6 expression positively correlates with NPM1 in PDAC tissues, and their concurrent high expression is significantly associated with poor clinical outcomes. This study characterizes CSN6 as an oncogenic protein that promotes NPM1 stabilization by interacting with DCAF1, thereby enhancing ribosome biogenesis and cellular resistance to gemcitabine in PDAC. NPM1 may serve as a therapeutic target for CSN6 high PDAC that exhibits gemcitabine drug resistance.

Antigen receptor diversity depends on the assembly of variable (V), diverse (D), and joining (J) exons in genes encoding immunoglobulins (Igs) and T cell receptors (TCRs). During V(D)J recombination, DNA double-strand breaks (DSBs) introduced by the RAG1/2 nuclease complex are repaired by the process of nonhomologous end-joining (NHEJ). We hypothesized that functional redundancies between NHEJ and the chromatin DSB response, which depends on the kinase ATM, potentially masked the activity of additional factors that regulate V(D)J recombination. We performed targeted CRISPR-Cas9 knockout screens for genes implicated in V(D)J recombination in pro-B cells that were either untreated or treated with an ATM inhibitor. We found that loss of the RNA/DNA helicase senataxin (SETX) impaired V(D)J recombination and led to the formation of aberrant hybrid joints between coding ends and signal ends, both in vitro and in mice. The loss of SETX in a background deficient in the NHEJ factor XLF or in which ATM was inhibited led to substantial impairment of V(D)J recombination and to the presence of unsealed coding ends. SETX limited aberrant activation-induced cytidine deaminase (AID)-induced DNA end-joining between Igh-containing alleles during the process of class-switch recombination. Together, our findings reveal a previously uncharacterized role for SETX in promoting recombination fidelity during antigen receptor gene diversification.

Protein kinase GIα oxidation negatively regulates antibody production by B cells

Cytosine base editors (CBEs) achieve precise C-to-T conversions by addition of uracil DNA glycosylase inhibitor (UGI) with Cas9 nickase (nCas9) and cytidine deaminase, and the conventional fusion at the nCas9 carboxyl terminus effectively inhibits uracil excision repair to enhance editing efficiency. However, despite potent on-target activity, classical CBEs exhibit significant Cas9-dependent DNA off-target effects that necessitate optimization for future applications. Here we present a strategic UGI relocation through internal fusion within the nCas9 architecture. This spatial reorganization maintains comparable on-target editing efficiency while substantially reducing Cas9-dependent DNA off-target activity. Our findings establish an alternative engineering paradigm to develop high-fidelity CBEs, offering an improved platform for widespread genome editing applications.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-19482-w.

Effects of Tetrahydrouridine Pre-dosing on Absorption, Metabolism, and Excretion of Decitabine in the Mouse.

Expression and characterization of activation-induced deaminase (AID) with dehydroaminase activity and its localization in IgM+ lymphocytes of spleen of common carp (Cyprinus carpio).

Engineering outer membrane vesicles (OMVs) carrying OmpA from S. Typhimurium for targeted modulation of human B-cell function through AID expression and class switch recombination.

Development of APOBEC3B inhibitors: Recent advances and perspectives.

Nuclear Translocation of Activation-Induced Cytidine Deaminase (AID) in ADPKD Tissue Reveals a Mechanism for Second Hit Mutations Leading to ADPKD

Tumor necrosis factor receptor-associated factors (TRAFs) are a family of adaptor proteins that transmit signals from immunoregulatory receptors—such as TNF receptors, Toll-like receptors, and interleukin receptors—to coordinate immune and inflammatory responses. Among them, TRAF5 is highly expressed in lymphocytes and implicated in obesity-associated inflammation, but its role in secondary lymphoid organs during chronic low-grade inflammation remains unclear. We examined splenic B and T cell phenotypes in wild-type (WT) and Traf5-deficient (KO) mice fed a high-fat diet (HFD). Although lymphocyte composition was broadly comparable, KO mice showed reduced spontaneous immunoglobulin G2c (IgG2c) production ex vivo—about 1.5-fold lower than WT. Notably, despite elevated TNF-α and CD40 ligand (CD40L) expression in HFD-fed KO splenocytes, IgG2c production remained diminished—about 1.9-fold lower than WT—upon soluble CD40L stimulation, indicating impaired CD40-mediated class-switch recombination (CSR). Consistently, B cells from KO mice on a normal diet exhibited reduced activation-induced cytidine deaminase (AID) expression—about 4.4-fold lower than WT—after CD40L stimulation, and decreased IgG2c secretion—about 6.6-fold lower—upon CD40L and IFN-γ co-stimulation in vitro. Collectively, these findings suggest that TRAF5 is involved in CD40-dependent CSR in B cells under inflammatory conditions and may contribute to sustaining adaptive immune responses during obesity-associated chronic inflammation.

Lichen sclerosus et atrophicus (LSA) is a chronic, progressive inflammatory disorder with a risk of malignant transformation to squamous cell carcinoma (SCC). This study investigated the role of activation-induced cytidine deaminase (AID) in UV-independent SCC arising from LSA. We retrospectively analyzed 19 female patients, including 5 SCC cases. AID expression was significantly associated with SCC development, pruritus, and shorter disease duration. AID was detected in SCC lesions and adjacent LSA tissues, mainly in basal cells. Reduced AID expression was observed in higher-stage tumors, suggesting its potential as a prognostic indicator. The use of topical corticosteroids was less common in AID-positive patients, suggesting a possible association. These findings suggest a possible inverse association between topical corticosteroid use and AID expression.

Structural insights into DdCBE in action enable high-precision mitochondrial DNA editing.

Apobec3 (A3, apolipoprotein B editing complex 3) is a cytidine deaminase with broadly antiretroviral activity. A3 is packaged in virions and converts cytidines to uracil (C > U) in viral minus-strand DNA during reverse transcription, resulting in guanosine to adenosine (G > A) mutations in the viral plus strand. Mouse Apobec3 (mA3) mutagenizes numerous infectious retroviruses and some families of endogenous retroviruses. Here we show that endogenous mouse mammary tumor viruses, termed Mtvs, show a range of mA3 hypermutation from trace levels to an extreme example, Mtv21, that sustained a catastrophic, genome-wide reduction of G content to 10.8%; this level is associated with the contemporaneous generation of new preferred trinucleotide target sites by mutagenesis of consecutive cytidines. Although newly inserted proviruses have identical long terminal repeats (LTRs), mA3 can disproportionately generate G > A substitutions specific to the Mtv 3' LTR. Individual hypermutated Mtvs display two different mA3 target site context preferences that correspond to those of the two mA3 alleles found among inbred strains of laboratory mice. Mtv-positive Mus musculus subspecies screened for these mA3 alleles unexpectedly revealed 20 distinct mA3 haplotypes with different combinations of replacement mutations at the 15 sites that distinguish the two inbred strain alleles, 11 of which show signatures of positive selection. These data show that mA3 deamination can have a major impact on the genomic integrity of Mtvs, and that mA3 is actively evolving within this single mouse species suggestive of high-impact genetic conflicts over a short evolutionary time frame.IMPORTANCEThe antiviral cytidine deaminase Apobec3 (apolipoprotein B editing complex 3) mutates retroviral DNA copies generated during new infections. Although such mutagenesis of replicating mouse retroviruses has been reported to be modest, here we show that many germline copies of mouse mammary tumor viruses (Mtvs) have sustained significant to massive levels of mouse Apobec3 (mA3) editing. mA3 hypermutation can also disproportionally affect one of the two otherwise identical viral long terminal repeats and can create new preferred target sites in the viral genome substrate by mutating successive cytosines. The edits in individual Mtvs correspond to the different target sequence preferences of the two inbred mouse strain mA3 alleles, but examination of allelic variation in wild mice of the species Mus musculus identified 18 additional variants and signatures of diversifying selection, a display of unusually rapid evolution within a single species over a short time frame.

Vaccination offers the most effective protection against contagious infectious diseases primarily by inducing humoral immunity. Vaccination efficacy is influenced by various factors. We report that dietary administration of arachidonic acid (ARA) significantly boosts rabies vaccine-induced production of neutralizing antibodies and protection against lethal rabies virus (RABV) infection in mice. In human volunteers, oral supplementation of ARA accelerates the expression of neutralizing antibodies to the levels sufficient for protection against RABV as early as one week after primary immunization. Mechanistically, ARA is enriched in lymph nodes and metabolized into immune modulators there. One of the ARA metabolites, prostaglandin I2 (PGI2), via the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) axis, upregulates the expression of costimulatory molecule CD86, and activates activation-induced cytidine deaminase (AID) in B cells. These results suggest that ARA can be a potent dietary adjuvant to foster germinal center (GC) B cell response and humoral immunity.

AID/HAT1-mediated epigenetic priming of JAG1/NOTCH signaling drives tumor microenvironment reprogramming in TNBC.

Class switch recombination (CSR) and locus suicide recombination (LSR) are critical processes involved in the immune system’s ability to diversify antibody responses. Both are initiated by activation-induced cytidine deaminase, which induces DNA double-strand breaks (DSBs) at specific regions within the immunoglobulin heavy chain (IgH) locus. In CSR, DSBs occur at the switch (S) regions, allowing B cells to replace the IgM heavy chain constant region (CH) with other isotypes, thereby enhancing immune adaptability. This process is regulated by both cis and trans mechanisms, including the IgH super-enhancer 3′ regulatory region (3′RR) and the production of enhancer RNAs (eRNAs). A recent study highlighted the role of MED12 in CSR through enhancer activation and the transcription of eRNA. Now, we show that heterogeneous ribonucleoprotein L (hnRNPL) acts as an additional regulator of CSR and LSR by forming an eRNA-associated complex with CstF64, a polyadenylation factor. This complex facilitates RNA polymerase II elongation and eRNA transcription at the 3′RR. Moreover, the hnRNPL/CstF64 complex promotes NHEJ-mediated DNA repair at both S and 3′RR regions, facilitating 53BP1 and Ku80 recruitment, thereby impacting the efficiency of CSR and LSR. This discovery highlights the intricate, multimodal regulation of these processes, linking eRNA transcription to DNA repair in the process of antibody diversification.

Gemcitabine is commonly used in the standard first-line treatment of urothelial carcinoma (UC); however, the emergence of drug resistance significantly limits its clinical benefit. The present study aims to investigate the role of CUB domain-containing protein 1 (CDCP1) in mediating resistance to gemcitabine in UC cells. Gemcitabine-resistant T24 (T24-GR) cells exhibited downregulation of human equilibrative nucleoside transporter 1 and upregulation of cytidine deaminase, key regulators of gemcitabine metabolism, as well as increased CDCP1 expression. Notably, silencing CDCP1 reversed these resistance-associated expression patterns. Mechanistically, T24-GR cells displayed elevated expression of CDCP1 and increased phosphorylation of c-Src and PKCδ, indicating activation of downstream survival signaling. Overexpression of CDCP1 in T24-CD cells activated similar pathways and modulated regulators of gemcitabine metabolism. In contrast, CRISPR/Cas9-mediated knockout of CDCP1 in T24-CDKO cells suppressed c-Src/PKCδ signaling and increased sensitivity to gemcitabine-induced cytotoxicity. Using flow cytometry, we observed that treatment with gemcitabine induced apoptosis in parental T24 cells, as indicated by an increase in the sub-G1 population. In contrast, T24-GR and T24-CD cells showed minimal sub-G1 accumulation, suggesting resistance to gemcitabine-induced apoptosis. Western blot analysis revealed decreased levels of cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase in T24-GR and T24-CD cells following gemcitabine exposure, whereas these markers were upregulated in parental T24 and T24-CDKO cells. Furthermore, the knockdown of CDCP1 and the utilization of c-Src/PKCδ signaling inhibitors in T24-GR cells led to the restoration of sensitivity to gemcitabine. By suppressing apoptosis and altering drug metabolism pathways, highlighting CDCP1 as a potential therapeutic target for overcoming gemcitabine resistance in UC.