The cytotoxic mechanisms of thymidylate synthase inhibitors, such as the multitarget antifolate pemetrexed, are not yet fully understood. Emerging evidence indicates that combining pemetrexed with histone deacetylase inhibitors (HDACi) may enhance therapeutic efficacy in non-small cell lung cancer (NSCLC). To explore this further, A549 NSCLC cells were treated with various combinations of pemetrexed and the HDACi MS275 (Entinostat), and subsequently assessed for cell viability, cell cycle changes, and genotoxic markers. Proteomic alterations were analyzed using label-free shotgun and targeted LC–MS/MS. MS275 enhanced the sensitivity of A549 cells to pemetrexed, but only when administered following prior treatment with pemetrexed. Both HeLa (p53 negative) and A549 (p53 positive) showed robust activation of γH2AX upon treatment with this combination. Importantly, CRISPR/Cas9 knockout of the uracil-DNA glycosylase UNG did not affect γH2AX activation or sensitivity to pemetrexed. Proteomic analysis revealed that MS275 altered the expression of known pemetrexed targets, as well as several proteins involved in pyrimidine metabolism and DNA repair, which could potentiate pemetrexed cytotoxicity. Contrary to the conventional model of antifolate toxicity, which implicates futile cycles of uracil incorporation and excision in DNA, we propose that ribonucleotide incorporation in nuclear and mitochondrial DNA significantly contributes to the cytotoxicity of antifolates like pemetrexed, and likely also of fluorinated pyrimidine analogs. HDAC inhibition apparently exacerbates cytotoxicity of these agents by inhibiting error-free repair of misincorporated ribonucleotides in DNA. The potential of HDACis to modulate pyrimidine metabolism and DNA damage responses offers novel strategies for improving NSCLC outcomes.

Activation-induced cytidine deaminase (AID) interacts with replication protein A (RPA), the major ssDNA-binding protein, to promote deamination of cytosine to uracil in transcribed immunoglobulin (Ig) genes. Uracil-DNA glycosylase (UNG) acts in concert with AID during Ig diversification. In addition, UNG preserves genome integrity by base-excision repair (BER) in the overall genome. How UNG is regulated to support both mutagenic processing and error-free repair remains unknown. UNG is expressed as two isoforms, UNG1 and UNG2, which both contain an RPA-binding helix that facilitates uracil excision from RPA-coated ssDNA. However, the impact of this interaction in antibody diversification and genome maintenance has not been investigated. Here, we generated B-cell clones with targeted mutations in the UNG RPA-binding motif, and analysed class switch recombination (CSR), mutation frequency (5' Ig Sμ), and genomic uracil in clones representing seven Ung genotypes. We show that the UNG:RPA interaction plays a crucial role in both CSR and repair of AID-induced uracil at the Ig loci. By contrast, the interaction had no significant impact on total genomic uracil levels. Thus, RPA coordinates UNG during CSR and pre-replicative repair of mutagenic uracil in ssDNA but is not essential in post-replicative and canonical BER of uracil in dsDNA.

Abstract Uracil occurs at replication forks via misincorporation of deoxyuridine monophosphate (dUMP) or via deamination of existing cytosines, which occurs 2–3 orders of magnitude faster in ssDNA than in dsDNA and is 100% miscoding. Tethering of UNG2 to proliferating cell nuclear antigen (PCNA) allows rapid post-replicative removal of misincorporated uracil, but potential ‘pre-replicative’ removal of deaminated cytosines in ssDNA has been questioned since this could mediate mutagenic translesion synthesis and induction of double-strand breaks. Here, we demonstrate that uracil-DNA glycosylase (UNG), but not SMUG1 efficiently excises uracil from replication protein A (RPA)-coated ssDNA and that this depends on functional interaction between the flexible winged-helix (WH) domain of RPA2 and the N-terminal RPA-binding helix in UNG. This functional interaction is promoted by mono-ubiquitination and diminished by cell-cycle regulated phosphorylations on UNG. Six other human proteins bind the RPA2-WH domain, all of which are involved in DNA repair and replication fork remodelling. Based on this and the recent discovery of the AP site crosslinking protein HMCES, we propose an integrated model in which templated repair of uracil and potentially other mutagenic base lesions in ssDNA at the replication fork, is orchestrated by RPA. The UNG:RPA2-WH interaction may also play a role in adaptive immunity by promoting efficient excision of AID-induced uracils in transcribed immunoglobulin loci.

U–G mismatches in DNA generally result from spontaneous cytosine deamination and are repaired by the base excision repair pathway to avoid mutations. However, as part of the adaptive immune response in B cells U–G mismatches are enzymatically generated in the Ig genes by activation-induced cytidine deaminase. These uracils are further processed by the uracil–DNA glycosylase UNG and funneled into mutagenic pathways to generate point mutations and strand breaks resulting in somatic hypermutation and class shift recombination, respectively. Cytosine deamination by activation-induced cytidine deaminase and other DNA deaminases is also a significant source of mutations in cancer. Human DNA polymerases do not discriminate between dTTP and dUTP. Misincorporation of dUMP generates U–A pairs that are indirectly mutagenic through erroneous processing of abasic sites. Some cells have surprisingly high dUTP/dTTP ratios, suggesting that the incorporation of dUMP during DNA replication or repair may be considerable. Mammalian cells have four different uracil–DNA glycosylases; UNG, SMUG1, TDG and MBD4. These have in part complementary, but also distinct functions in base excision repair, immunity and epigenetic regulation. In addition, they may modify responses to fluoropyrimidines in cancer treatment.

To ensure genome stability, mammalian cells employ several DNA repair pathways. Nonhomologous DNA end joining ( NHEJ ) is the DNA repair process that fixes double‐strand breaks throughout the cell cycle. NHEJ is involved in the development of B and T lymphocytes through its function in V(D)J recombination and class switch recombination ( CSR ). NHEJ consists of several core and accessory factors, including Ku70, Ku80, XRCC 4, DNA ligase 4, DNA ‐ PK cs, Artemis, and XLF . Paralog of XRCC 4 and XLF ( PAXX ) is the recently described accessory NHEJ factor that structurally resembles XRCC 4 and XLF and interacts with Ku70/Ku80. To determine the physiological role of PAXX in mammalian cells, we purchased and characterized a set of custom‐generated and commercially available NHEJ ‐deficient human haploid HAP 1 cells, PAXX Δ , XRCC 4 Δ , and XLF Δ . In our studies, HAP 1 PAXX Δ cells demonstrated modest sensitivity to DNA damage, which was comparable to wild‐type controls. By contrast, XRCC 4 Δ and XLF Δ HAP 1 cells possessed significant DNA repair defects measured as sensitivity to double‐strand break inducing agents and chromosomal breaks. To investigate the role of PAXX in CSR , we generated and characterized Paxx −/− and Aid −/− murine lymphoid CH 12F3 cells. CSR to IgA was nearly at wild‐type levels in the Paxx −/− cells and completely ablated in the absence of activation‐induced cytidine deaminase ( AID ). In addition, Paxx −/− CH 12F3 cells were hypersensitive to zeocin when compared to wild‐type controls. We concluded that Paxx ‐deficient mammalian cells maintain robust NHEJ and CSR .

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, pp. 1-13 (2018) Free AccessChapter 1: Introduction — DNA Repair is Integrated with Many Cellular ProcessesHans E. Krokan, Bodil Kavli and Geir SlupphaugHans E. Krokan, Bodil Kavli and Geir Slupphaughttps://doi.org/10.1142/9789813233508_0001Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: Long-term survival of a species requires cellular mechanisms that efficiently safeguard DNA, the chemical material of the genome. All known living organisms have several mechanisms that repair different types of DNA damage. This is essential to prevent cytotoxic and mutagenic effects of damage to DNA. In fact, each cell in the body is inflicted by tens of thousands of DNA lesions per day from spontaneous chemical decay and normal cellular metabolites alone. Environmental chemicals, ultraviolet and ionizing radiation add to the burden of genome damage. Without cellular mechanisms that maintain DNA, we would probably die within a few days from multiple organ failure due to inactivation of numerous genes in each cell. Fortunately, a number of mechanisms contribute to maintaining DNA, including high fidelity DNA replication, DNA repair, cell cycle regulation, as well as removal of cells with serious genetic defects by apoptosis (programmed cell death) (Fig. 1.1). However, this is just the tip of the iceberg, as outlined below. FiguresReferencesRelatedDetails Genomic UracilMetrics Downloaded 200 times History PDF download

Genomic Uracil, pp. 127-152 (2018) No AccessChapter 5: Viral Uracil — Uracil DNA Glycosylases and dUTPasesHans E. Krokan, Bodil Kavli, and Geir SlupphaugHans E. Krokan, Bodil Kavli, and Geir Slupphaughttps://doi.org/10.1142/9789813233508_0005Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: DNA viruses and retroviruses use various strategies to avoid and remove genomic uracil. Similar to the case for cells, dUTPase and UNG-type uracil-DNA glycosylase are central in these processes. Large DNA viruses encode viral variants of Family 1 (UNG) enzymes, while smaller viruses depend solely on host cell enzymes. Genes for viral dUTPase and UNG are among those that are expressed early after infection. Herpesviridae and poxviridae encode their own uracil-DNA glycosylase, dUTPase and DNA polymerase. Midsize DNA viruses in the adenoviridae family encode their own DNA polymerase, but not uracil-DNA glycosylase. Fowl adenovirus 9 also encodes a dUTPase, but in human adenoviruses a dUTPase-like gene has diverged to become a transforming factor named E4-ORF1. Retroviridae do not encode uracil-DNA glycosylase, but some of them capture host uracil-DNA glycosylase, which is encapsulated in the virus particle and released after infection. Furthermore, several major retroviruses encode dUTPase, although this is not the case for primate lentiviruses that instead use captured UNG to minimize genomic uracil in replicative DNA intermediates. Herpesvirus uracil-DNA glycosylase has strong homology to E. coli, yeast and mammalian UNG proteins, while Poxvirus uracil-DNA glycosylase is a more distant member of the same family. Herpesviruses and adenoviruses replicate in the host nucleus, where they in part can rely on host factors. In contrast, poxviruses replicate in replication factories in the cytosol and encode several DNA replication and repair proteins as well as nucleotide metabolizing enzymes. Interestingly, while herpesvirus and poxvirus UNG proteins are genuine uracil-DNA glycosylases, they also have important functions in viral replication. This has been extensively studied for poxvirus UNG, which is essential for virus replication. Poxvirus UNG (D4) forms a heterotrimeric complex with an adapter protein (A20) and the pox DNA polymerase (E9) and acts as a polymerase processivity factor. Herpes UNG is particularly important for virulence in nerve cells, which lack or have very low levels of cellular UNG. The function of host UNG in retroviridae is complex, as outlined below. The special functions of viral UNG and dUTPase make these proteins potential drug targets. The giant mimivirus Acanthamoeba polyphaga (see Chapter 2) encodes numerous DNA repair proteins, including an UNG-type uracil-DNA glycosylase. Its sequence and crystal structure demonstrate the presence of a catalytic domain containing structural and functional motifs typically present in other UNG proteins and an unstructured N-terminal extension typical of mammalian UNG proteins. FiguresReferencesRelatedDetails Genomic UracilMetrics History PDF download

Genomic Uracil, pp. 183-244 (2018) No AccessChapter 7: Genomic Uracil and CancerAntonio Sarno, Pål Sætrom, Bodil Kavli, and Henrik Sahlin PettersenAntonio Sarno, Pål Sætrom, Bodil Kavli, and Henrik Sahlin Pettersenhttps://doi.org/10.1142/9789813233508_0007Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: New technologies have made possible sequencing of a large number of whole cancer genomes and exomes. Such studies have identified sequence-associated mutational signatures in human cancers. These frequently suggest a causative mutational agent, thus shedding light on the molecular mechanisms of oncogenesis and cancer progression. Analysis of mutational signatures may also be used to monitor response to therapy… FiguresReferencesRelatedDetails Genomic UracilMetrics History PDF download

Genomic Uracil, pp. 153-182 (2018) No AccessChapter 6: Genomic Uracil and ImmunityBodil Kavli and Geir SlupphaugBodil Kavli and Geir Slupphaughttps://doi.org/10.1142/9789813233508_0006Cited by:0 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: Identification of uracil in DNA as a key intermediate in both adaptive and innate immunity has made research on the introduction and fate of this non-canonical DNA base essential for understanding how we defend ourselves against invading pathogenic microorganisms. All cells have repair systems that remove DNA lesions to preserve genetic integrity. Occasionally, however, some lesions escape repair and result in changes in the DNA sequence. Together with selection of the fittest, this is the concept of evolution of all species. To combat pathogenic bacteria and virus (antigens), specific immune cells (B lymphocytes/B cells) have developed a sophisticated high-speed gene-targeted evolutionary process, termed somatic hypermutation (SHM). This extraordinary mechanism makes B cells capable of producing "tailor-made" high affinity antibodies (immunoglobulins) against any antigen. To initiate SHM, activated B-cells express an enzyme; activation-induced deaminase (AID) that introduces uracil in specific regions of the immunoglobulin (Ig) genes. These uracils are generally not repaired by error free BER, but are processed to generate point mutations or double strand breaks that modulate the antibody binding site or initiate Ig isotype switching, respectively. Cells expressing membrane-bound Igs (B-cell receptors) with increased affinity for antigens are selected to proliferate and differentiate into antibody-secreting plasma cells and memory cells, which combat current infections and protect us against similar future pathogens… FiguresReferencesRelatedDetails Genomic UracilMetrics History PDF download

Genomic Uracil, pp. 89-126 (2018) No AccessChapter 4: Enzymology of Genomic Uracil RepairHans E. Krokan, Bodil Kavli, Antonio Sarno, and Geir SlupphaugHans E. Krokan, Bodil Kavli, Antonio Sarno, and Geir Slupphaughttps://doi.org/10.1142/9789813233508_0004Cited by:2 PreviousNext AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsRecommend to Library ShareShare onFacebookTwitterLinked InRedditEmail Abstract: Whereas several laboratories contributed to the discovery of other DNA excision repair processes, the discovery of base excision repair (BER) can be uniquely ascribed to the work of Tomas Lindahl (Fig. 4.1). He quantified spontaneous deamination of cytosine to uracil in genomic DNA and reasoned that there had to be a mecshanism correcting the highly mutagenic U:G mismatches resulting from the deamination. The search for such a mechanism resulted in the surprising discovery of uracil-DNA glycosylase in 1974, the first enzyme in a new family of DNA repair proteins. The new enzyme was initially named 'uracil N-glycosidase,' but the name was later changed to uracil-DNA glycosylase to comply with current nomenclature. In addition to his work on DNA-cytosine deamination, Lindahl made important discoveries on other aspects of DNA instability, including rates of depurination, depyrimidination and chain breaks at abasic sites… FiguresReferencesRelatedDetailsCited By 2Base Excision Repair in Sugarcane – A New OutlookNathalia Maíra Cabral de Medeiros and Katia Castanho Scortecci23 June 2021HDACi mediate UNG2 depletion, dysregulated genomic uracil and altered expression of oncoproteins and tumor suppressors in B- and T-cell linesTobias S. Iveland, Lars Hagen, Animesh Sharma, Mirta M. L. Sousa and Antonio Sarno et al.7 April 2020 | Journal of Translational Medicine, Vol. 18, No. 1 Genomic UracilMetrics History PDF download

Human uracil N-glycosylase isoform 2—UNG2 consists of an N-terminal intrinsically disordered regulatory domain (UNG2 residues 1–92, 9.3 kDa) and a C-terminal structured catalytic domain (UNG2 residues 93–313, 25.1 kDa). Here, we report the backbone 1H, 13C, and 15N chemical shift assignment as well as secondary structure analysis of the N-and C-terminal domains of UNG2 representing the full-length UNG2 protein.

Genes encoding pectic enzymes were introduced to wild-type potato Karnico. Cell wall materials were extracted from Karnico and transgenic lines expressing β-galactosidase (β-Gal-14 mutant) or rhamnogalacturonan lyase (RGL-18 mutant). After sequential extraction, β-Gal-14 hot buffer-soluble solids (HBSS) of pectin contained 54% less galactose than Karnico HBSS, representing shorter galactan side chains. The individual pectin populations of β-Gal-14 HBSS showed different modifications extended to the two sub-populations as obtained by ion-exchange chromatography. Compared to wild-type, RGL-18 HBSS contained 27% more galacturonic acid and 55% less Gal on fresh potato weight basis, which was due to the removal of galactan-rich rhamnogalacturonan I (RG-I) segments. All pectin populations of RGL-18 showed consistently low levels of RG-I segments. Transgenic modification showed side effects on the methyl-esterification and acetyl substitution of RGL-18 HBSS (DM = 53, DA = 21), but not of the β-Gal-14 HBSS in comparison to wild-type (DM = 29, DA = 54).

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.

Activation-induced cytidine deaminase (AID) is the mutator enzyme in adaptive immunity. AID initiates the antibody diversification processes in activated B cells by deaminating cytosine to uracil in immunoglobulin genes. To some extent other genes are also targeted, which may lead to genome instability and B cell malignancy. Thus, it is crucial to understand its targeting and regulation mechanisms. AID is regulated at several levels including subcellular compartmentalization. However, the complex nuclear distribution and trafficking of AID has not been studied in detail previously. In this work, we examined the subnuclear localization of AID and its interaction partner CTNNBL1 and found that they associate with spliceosome-associated structures including Cajal bodies and nuclear speckles. Moreover, protein kinase A (PKA), which activates AID by phosphorylation at Ser38, is present together with AID in nuclear speckles. Importantly, we demonstrate that AID physically associates with the major spliceosome subunits (small nuclear ribonucleoproteins, snRNPs), as well as other essential splicing components, in addition to the transcription machinery. Based on our findings and the literature, we suggest a transcription-coupled splicing-associated model for AID targeting and activation.

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.

Genomic uracil is normally processed essentially error-free by base excision repair (BER), with mismatch repair (MMR) as an apparent backup for U:G mismatches. Nuclear uracil-DNA glycosylase UNG2 is the major enzyme initiating BER of uracil of U:A pairs as well as U:G mismatches. Deficiency in UNG2 results in several-fold increases in genomic uracil in mammalian cells. Thus, the alternative uracil-removing glycosylases, SMUG1, TDG and MBD4 cannot efficiently complement UNG2-deficiency. A major function of SMUG1 is probably to remove 5-hydroxymethyluracil from DNA with general back-up for UNG2 as a minor function. TDG and MBD4 remove deamination products U or T mismatched to G in CpG/mCpG contexts, but may have equally or more important functions in development, epigenetics and gene regulation. Genomic uracil was previously thought to arise only from spontaneous cytosine deamination and incorporation of dUMP, generating U:G mismatches and U:A pairs, respectively. However, the identification of activation-induced cytidine deaminase (AID) and other APOBEC family members as DNA-cytosine deaminases has spurred renewed interest in the processing of genomic uracil. Importantly, AID triggers the adaptive immune response involving error-prone processing of U:G mismatches, but also contributes to B-cell lymphomagenesis. Furthermore, mutational signatures in a substantial fraction of other human cancers are consistent with APOBEC-induced mutagenesis, with U:G mismatches as prime suspects. Mutations can be caused by replicative polymerases copying uracil in U:G mismatches, or by translesion polymerases that insert incorrect bases opposite abasic sites after uracil-removal. In addition, kataegis, localized hypermutations in one strand in the vicinity of genomic rearrangements, requires APOBEC protein, UNG2 and translesion polymerase REV1. What mechanisms govern error-free versus error prone processing of uracil in DNA remains unclear. In conclusion, genomic uracil is an essential intermediate in adaptive immunity and innate antiviral responses, but may also be a fundamental cause of a wide range of malignancies.