UNG is the major uracil-DNA glycosylase in mammalian cells and is involved in both error-free base excision repair of genomic uracil and mutagenic uracil-processing at the antibody genes. However, the regulation of UNG in these different processes is currently not well understood. The UNG gene encodes two isoforms, UNG1 and UNG2, each possessing unique N-termini that mediate translocation to the mitochondria and the nucleus, respectively. A strict subcellular localization of each isoform has been widely accepted despite a lack of models to study them individually. To determine the roles of each isoform, we generated and characterized several UNG isoform-specific mouse and human cell lines. We identified a distinct UNG1 isoform variant that is targeted to the cell nucleus where it supports antibody class switching and repairs genomic uracil. We propose that the nuclear UNG1 variant, which in contrast to UNG2 lacks a PCNA-binding motif, may be specialized to act on ssDNA through its ability to bind RPA. RPA-coated ssDNA regions include both transcribed antibody genes that are targets for deamination by AID and regions in front of the moving replication forks. Our findings provide new insights into the function of UNG isoforms in adaptive immunity and DNA repair.

Genomic uracil resulting from spontaneously deaminated cytosine generates mutagenic U:G mismatches that are usually corrected by error-free base excision repair (BER). However, in B-cells, activation-induced cytosine deaminase (AID) generates U:G mismatches in hot-spot sequences at Ig loci. These are subject to mutagenic processing during somatic hypermutation (SHM) and class switch recombination (CSR). Uracil N-glycosylases UNG2 and SMUG1 (single strand-selective monofunctional uracil-DNA glycosylase 1) initiate error-free BER in most DNA contexts, but UNG2 is also involved in mutagenic processing of AID-induced uracil during the antibody diversification process, the regulation of which is not understood. AID is strictly single strand-specific. Here we show that in the presence of Mg2+ and monovalent salts, human and mouse SMUG1 are essentially double strand-specific, whereas UNG2 efficiently removes uracil from both single and double stranded DNA under all tested conditions. Furthermore, SMUG1 and UNG2 display widely different sequence preferences. Interestingly, uracil in a hot-spot sequence for AID is 200-fold more efficiently removed from single stranded DNA by UNG2 than by SMUG1. This may explain why SMUG1, which is not excluded from Ig loci, is unable to replace UNG2 in antibody diversification. We suggest a model for mutagenic processing in which replication protein A (RPA) recruits UNG2 to sites of deamination and keeps DNA in a single stranded conformation, thus avoiding error-free BER of the deaminated cytosine.

The generation of high-affinity antibodies requires somatic hypermutation (SHM) and class switch recombination (CSR) at the immunoglobulin (Ig) locus. Both processes are triggered by activation-induced cytidine deaminase (AID) and require UNG-encoded uracil-DNA glycosylase. AID has been suggested to function as an mRNA editing deaminase or as a single-strand DNA deaminase. In the latter model, SHM may result from replicative incorporation of dAMP opposite U or from error-prone repair of U, whereas CSR may be triggered by strand breaks at abasic sites. Here, we demonstrate that extracts of UNG-proficient human B cell lines efficiently remove U from single-stranded DNA. In B cell lines from hyper-IgM patients carrying UNG mutations, the single-strand-specific uracil-DNA glycosylase, SMUG1, cannot complement this function. Moreover, the UNG mutations lead to increased accumulation of genomic uracil. One mutation results in an F251S substitution in the UNG catalytic domain. Although this UNG form was fully active and stable when expressed in Escherichia coli, it was mistargeted to mitochondria and degraded in mammalian cells. Our results may explain why SMUG1 cannot compensate the UNG2 deficiency in human B cells, and are fully consistent with the DNA deamination model that requires active nuclear UNG2. Based on our findings and recent information in the literature, we present an integrated model for the initiating steps in CSR.

B-lymphocytes can modify their immunoglobulin (Ig) genes to generate specific antibodies with a new isotype and enhanced affinity against an antigen. Activation-induced cytidine deaminase (AID), which is positively regulated by the transcription factor E2A, is the key enzyme that initiates these processes by deaminating cytosine to uracil in Ig genes. Nuclear uracil-DNA glycosylase (UNG2) is subsequently required for uracil processing in the generation of high affinity antibodies of different isotypes. Here we show that the transcription factor E2A binds to the UNG2 promoter and represses UNG2 expression. Inhibition of E2A by binding of Ca(2+)-activated calmodulin alleviates this repression. Furthermore, we demonstrate that UNG2 preferentially accumulates in regions of the Ig heavy chain (IgH) gene containing AID hotspots. Calmodulin inhibition of E2A strongly enhances this UNG2 accumulation, indicating that it is negatively regulated by E2A as well. We show also that over-expression of E2A can suppress class switch recombination. The results suggest that E2A is a key factor in regulating the balance between AID and UNG2, both at expression and Ig targeting levels, to stimulate Ig diversification and suppress normal DNA repair processes.

The most common mutations in cancer are C to T transitions, but their origin has remained elusive. Recently, mutational signatures of APOBEC-family cytosine deaminases were identified in many common cancers, suggesting off-target deamination of cytosine to uracil as a common mutagenic mechanism. Here we present evidence from mass spectrometric quantitation of deoxyuridine in DNA that shows significantly higher genomic uracil content in B-cell lymphoma cell lines compared to non-lymphoma cancer cell lines and normal circulating lymphocytes. The genomic uracil levels were highly correlated with AID mRNA and protein expression, but not with expression of other APOBECs. Accordingly, AID knockdown significantly reduced genomic uracil content. B-cells stimulated to express endogenous AID and undergo class switch recombination displayed a several-fold increase in total genomic uracil, indicating that B cells may undergo widespread cytosine deamination after stimulation. In line with this, we found that clustered mutations (kataegis) in lymphoma and chronic lymphocytic leukemia predominantly carry AID-hotspot mutational signatures. Moreover, we observed an inverse correlation of genomic uracil with uracil excision activity and expression of the uracil-DNA glycosylases UNG and SMUG1. In conclusion, AID-induced mutagenic U:G mismatches in DNA may be a fundamental and common cause of mutations in B-cell malignancies.

Both a DNA lesion and an intermediate for antibody maturation, uracil is primarily processed by base excision repair (BER), either initiated by uracil-DNA glycosylase (UNG) or by single-strand selective monofunctional uracil DNA glycosylase (SMUG1). The relative in vivo contributions of each glycosylase remain elusive. To assess the impact of SMUG1 deficiency, we measured uracil and 5-hydroxymethyluracil, another SMUG1 substrate, in Smug1 -/- mice. We found that 5-hydroxymethyluracil accumulated in Smug1 -/- tissues and correlated with 5-hydroxymethylcytosine levels. The highest increase was found in brain, which contained about 26-fold higher genomic 5-hydroxymethyluracil levels than the wild type. Smug1 -/- mice did not accumulate uracil in their genome and Ung -/- mice showed slightly elevated uracil levels. Contrastingly, Ung -/- Smug1 -/- mice showed a synergistic increase in uracil levels with up to 25-fold higher uracil levels than wild type. Whole genome sequencing of UNG/SMUG1-deficient tumours revealed that combined UNG and SMUG1 deficiency leads to the accumulation of mutations, primarily C to T transitions within CpG sequences. This unexpected sequence bias suggests that CpG dinucleotides are intrinsically more mutation prone. In conclusion, we showed that SMUG1 efficiently prevent genomic uracil accumulation, even in the presence of UNG, and identified mutational signatures associated with combined UNG and SMUG1 deficiency.

Ung-deficient mice have reduced class switch recombination, skewed somatic hypermutation, lymphatic hyperplasia and a 22-fold increased risk of developing B-cell lymphomas. We find that lymphomas are of follicular (FL) and diffuse large B-cell type (DLBCL). All FLs and 75% of the DLBCLs were monoclonal while 25% were biclonal. Monoclonality was also observed in hyperplasia, and could represent an early stage of lymphoma development. Lymphoid hyperplasia occurs very early in otherwise healthy Ung-deficient mice, observed as a significant increase of splenic B-cells. Furthermore, loss of Ung also causes a significant reduction of T-helper cells, and 50% of the young Ung(-/-) mice investigated have no detectable NK/NKT-cell population in their spleen. The immunological imbalance is confirmed in experiments with spleen cells where the production of the cytokines interferon gamma, interleukin 6 and interleukin 2 is clearly different in wild type and in Ung-deficient mice. This suggests that Ung-proteins, directly or indirectly, have important functions in the immune system, not only in the process of antibody maturation, but also for production and functions of immunologically important cell types. The immunological imbalances shown here in the Ung-deficient mice may be central in the development of lymphomas in a background of generalised lymphoid hyperplasia.

Human UNG2 is a multifunctional glycosylase that removes uracil near replication forks and in non-replicating DNA, and is important for affinity maturation of antibodies in B cells. How these diverse functions are regulated remains obscure. Here, we report three new phosphoforms of the non-catalytic domain that confer distinct functional properties to UNG2. These are apparently generated by cyclin-dependent kinases through stepwise phosphorylation of S23, T60 and S64 in the cell cycle. Phosphorylation of S23 in late G1/early S confers increased association with replication protein A (RPA) and replicating chromatin and markedly increases the catalytic turnover of UNG2. Conversely, progressive phosphorylation of T60 and S64 throughout S phase mediates reduced binding to RPA and flag UNG2 for breakdown in G2 by forming a cyclin E/c-myc-like phosphodegron. The enhanced catalytic turnover of UNG2 p-S23 likely optimises the protein to excise uracil along with rapidly moving replication forks. Our findings may aid further studies of how UNG2 initiates mutagenic rather than repair processing of activation-induced deaminase-generated uracil at Ig loci in B cells.

Cytotoxicity of 5-fluorouracil (FU) and 5-fluoro-2'-deoxyuridine (FdUrd) due to DNA fragmentation during DNA repair has been proposed as an alternative to effects from thymidylate synthase (TS) inhibition or RNA incorporation. The goal of the present study was to investigate the relative contribution of the proposed mechanisms for cytotoxicity of 5-fluoropyrimidines. We demonstrate that in human cancer cells, base excision repair (BER) initiated by the uracil-DNA glycosylase UNG is the major route for FU-DNA repair in vitro and in vivo. SMUG1, TDG and MBD4 contributed modestly in vitro and not detectably in vivo. Contribution from mismatch repair was limited to FU:G contexts at best. Surprisingly, knockdown of individual uracil-DNA glycosylases or MSH2 did not affect sensitivity to FU or FdUrd. Inhibitors of common steps of BER or DNA damage signalling affected sensitivity to FdUrd and HmdUrd, but not to FU. In support of predominantly RNA-mediated cytotoxicity, FU-treated cells accumulated ~3000- to 15 000-fold more FU in RNA than in DNA. Moreover, FU-cytotoxicity was partially reversed by ribonucleosides, but not deoxyribonucleosides and FU displayed modest TS-inhibition compared to FdUrd. In conclusion, UNG-initiated BER is the major route for FU-DNA repair, but cytotoxicity of FU is predominantly RNA-mediated, while DNA-mediated effects are limited to FdUrd.

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.

Activation-induced cytidine deaminase (AID) is a DNA mutator enzyme essential for adaptive immunity. AID initiates somatic hypermutation and class switch recombination (CSR) by deaminating cytosine to uracil in specific immunoglobulin (Ig) gene regions. However, other loci, including cancer-related genes, are also targeted. Thus, tight regulation of AID is crucial to balance immunity versus disease such as cancer. AID is regulated by several mechanisms including nucleocytoplasmic shuttling. Here we have studied nuclear import kinetics and subnuclear trafficking of AID in live cells and characterized in detail its nuclear localization signal. Importantly, we find that the nuclear localization signal motif also directs AID to nucleoli where it colocalizes with its interaction partner, catenin-β-like 1 (CTNNBL1), and physically associates with nucleolin and nucleophosmin. Moreover, we demonstrate that release of AID from nucleoli is dependent on its C-terminal motif. Finally, we find that CSR efficiency correlates strongly with the arithmetic product of AID nuclear import rate and DNA deamination activity. Our findings suggest that directional nucleolar transit is important for the physiological function of AID and demonstrate that nuclear/nucleolar import and DNA cytosine deamination together define the biological activity of AID. This is the first study on subnuclear trafficking of AID and demonstrates a new level in its complex regulation. In addition, our results resolve the problem related to dissociation of deamination activity and CSR activity of AID mutants.