Deamination Of Cytosine In DNA Research Articles

Abstract PO5-27-03: Mutational impact of APOBEC3A and APOBEC3B in a human cell line

Abstract A major source of mutation in cancer is DNA cytosine deamination by APOBEC3 enzymes resulting in C-to-T and C-to-G mutations in TCA and TCT motifs. Here, we develop a selectable system to quantify genomic mutations and compare the mutagenic activities of three leading APOBEC3 candidates - APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and nearly indistinguishable TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from TK mutant clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs over 70% of the time and APOBEC3B just under 50% of the time (Y=C/T; W=A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes are contributing in a combinatorial manner to the overall mutation landscape. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both enzymes. Citation Format: Michael Carpenter, Nuri Temiz, Mahmoud Ibrahim, Matthew Jarvis, Margaret Brown, Prokopios Argyris, William Brown, Douglas Yee, Reuben Harris. Mutational impact of APOBEC3A and APOBEC3B in a human cell line [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-27-03.

Mutational impact of APOBEC3A and APOBEC3B in a human cell line and comparisons to breast cancer.

A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in signature C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which protein(s) are responsible. Here we report the development of a selectable system to quantify genome mutation and demonstrate its utility by comparing the mutagenic activities of three leading candidates-APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, is engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggers increased frequencies of TK mutation and similar TC-biased cytosine mutation profiles in the selectable TK reporter gene. Whole genome sequences from independent clones enabled an analysis of thousands of single base substitution mutations and extraction of local sequence preferences with APOBEC3A preferring YTCW motifs 70% of the time and APOBEC3B 50% of the time (Y = C/T; W = A/T). Signature comparisons with breast tumor whole genome sequences indicate that most malignancies manifest intermediate percentages of APOBEC3 signature mutations in YTCW motifs, mostly between 50 and 70%, suggesting that both enzymes contribute in a combinatorial manner to the overall mutation landscape. Although the vast majority of APOBEC3A- and APOBEC3B-induced single base substitution mutations occur outside of predicted chromosomal DNA hairpin structures, whole genome sequence analyses and supporting biochemical studies also indicate that both enzymes are capable of deaminating the single-stranded loop regions of DNA hairpins at elevated rates. These studies combine to help resolve a long-standing etiologic debate on the source of APOBEC3 signature mutations in cancer and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.

DNA cytosine deamination is associated with recurrent Somatic Copy Number Alterations in stomach adenocarcinoma.

Stomach Adenocarcinoma (STAD) is a leading cause of death worldwide. Somatic Copy Number Alterations (SCNAs), which result in Homologous recombination (HR) deficiency in double-strand break repair, are associated with the progression of STAD. However, the landscape of frequent breakpoints of SCNAs (hotspots) and their functional impacts remain poorly understood. In this study, we aimed to explore the frequency and impact of these hotspots in 332 STAD patients and 1,043 cancer cells using data from the Cancer Genome Atlas (TCGA) and Cancer Cell Line Encyclopedia (CCLE). We studied the rates of DSB (Double-Strand Breaks) loci in STAD patients by employing the Non-Homogeneous Poisson Distribution (λ), based on which we identified 145 DSB-hotspots with genes affected. We further verified DNA cytosine deamination as a critical process underlying the burden of DSB in STAD. Finally, we illustrated the clinical impact of the significant biological processes. Our findings highlighted the relationship between DNA cytosine deamination and SCNA in cancer was associated with recurrent Somatic Copy Number Alterations in STAD.

APOBEC3A Catalytic Inactivity Mutation Induces Tertiary Structure Destabilization.

APOBEC3A (A3A)-catalyzed DNA cytosine deamination is implicated in virus and cancer mutagenesis, and A3A is a target for small molecule drug discovery. The catalytic glutamic acid (E72) is frequently mutated in biochemical studies to characterize deamination-dependent versus deamination-independent A3A functions. Additionally, catalytically active A3A is toxic in bacterial expression systems, which adversely affects yield during recombinant A3A expression. Here, we demonstrate that mutating the catalytic glutamic acid to an isosteric glutamine (E72Q) significantly decreases the thermal stability of the protein, compared to the alanine-inactivating mutation (E72A). Differential scanning fluorimetry and mass spectrometry reveal that A3A E72Q is less thermally stable than A3A E72A or wild-type A3A. Strikingly, A3A E72Q is partially denatured at 37 °C and binds single-stranded DNA with significantly poorer affinity compared to A3A E72A. This study constitutes an important cautionary note on A3A protein design and informs that A3A E72A is the preferred catalytic inactivation mutation for most applications.

Genomic Uracil and Aberrant Profile of Demethylation Intermediates in Epigenetics and Hematologic Malignancies.

DNA of all living cells undergoes continuous structural and chemical alterations resulting from fundamental cellular metabolic processes and reactivity of normal cellular metabolites and constituents. Examples include enzymatically oxidized bases, aberrantly methylated bases, and deaminated bases, the latter largely uracil from deaminated cytosine. In addition, the non-canonical DNA base uracil may result from misincorporated dUMP. Furthermore, uracil generated by deamination of cytosine in DNA is not always damage as it is also an intermediate in normal somatic hypermutation (SHM) and class shift recombination (CSR) at the Ig locus of B-cells in adaptive immunity. Many of the modifications alter base-pairing properties and may thus cause replicative and transcriptional mutagenesis. The best known and most studied epigenetic mark in DNA is 5-methylcytosine (5mC), generated by a methyltransferase that uses SAM as methyl donor, usually in CpG contexts. Oxidation products of 5mC are now thought to be intermediates in active demethylation as well as epigenetic marks in their own rights. The aim of this review is to describe the endogenous processes that surround the generation and removal of the most common types of DNA nucleobase modifications, namely, uracil and certain epigenetic modifications, together with their role in the development of hematological malignances. We also discuss what dictates whether the presence of an altered nucleobase is defined as damage or a natural modification.

Abstract SP057: Mechanisms Controlling Expression of the Breast Cancer Genomic DNA Deaminase APOBEC3B

Abstract Introduction: APOBEC3B (A3B)-catalyzed DNA cytosine deamination contributes to the overall mutational landscape in primary and metastatic breast cancer. While low in normal tissues, A3B expression and activity are elevated in many primary tumors and increase further in metastases. Prior studies have implicated the RB/E2F signaling pathway in A3B overexpression in breast cancer but a detailed molecular mechanism has yet to be described. Methods: A combination of genetic, biochemical, proteomic, and bioinformatic approaches were used to investigate whether dysregulation of the RB/E2F signaling pathway is a molecular trigger for A3B overexpression in breast cancer. Results: First, an A3B promoter-driven luciferase reporter was used to demonstrate functional repression by one of five predicted E2F binding sites. Disruption of this single E2F site both phenocopied and prevented further induction by BK polyomavirus T antigen, which was known from prior studies to induce A3B expression. Second, targeted disruption of the endogenous E2F binding site caused strong A3B upregulation and confirmed the importance of this cis-regulatory element. Third, proteomics experiments showed that members of the DREAM and PRC1.6 complexes are able to bind to wildtype but not to mutant E2F promoter sequences. Additional biochemical, genetic, and cell biological studies implicated both the E2F4/DREAM complex and E2F6/PRC1.6 complex in endogenous A3B repression. Last, bioinformatic analyses of breast cancer data sets showed A3B overexpression and higher levels of APOBEC signature mutations in tumors with coordinately overexpressed E2F target genes. Conclusions: These studies combine to demonstrate that A3B expression is suppressed in normal cells by repressive E2F complexes and that disruption of this regulatory network triggers overexpression in breast cancer and provides fuel for tumor evolution. These studies raise the possibility that overexpression of an E2F gene set in breast tumors may be a biomarker for APOBEC mutability. Citation Format: R Harris. Mechanisms Controlling Expression of the Breast Cancer Genomic DNA Deaminase APOBEC3B [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr SP057.

Characterization of the mechanism by which the RB/E2F pathway controls expression of the cancer genomic DNA deaminase APOBEC3B.

APOBEC3B (A3B)-catalyzed DNA cytosine deamination contributes to the overall mutational landscape in breast cancer. Molecular mechanisms responsible for A3B upregulation in cancer are poorly understood. Here we show that a single E2F cis-element mediates repression in normal cells and that expression is activated by its mutational disruption in a reporter construct or the endogenous A3B gene. The same E2F site is required for A3B induction by polyomavirus T antigen indicating a shared molecular mechanism. Proteomic and biochemical experiments demonstrate the binding of wildtype but not mutant E2F promoters by repressive PRC1.6/E2F6 and DREAM/E2F4 complexes. Knockdown and overexpression studies confirm the involvement of these repressive complexes in regulating A3B expression. Altogether, these studies demonstrate that A3B expression is suppressed in normal cells by repressive E2F complexes and that viral or mutational disruption of this regulatory network triggers overexpression in breast cancer and provides fuel for tumor evolution.

APOBEC3B lysine residues are dispensable for DNA cytosine deamination, HIV-1 restriction, and nuclear localization

The APOBEC3 DNA cytosine deaminase family comprises a fundamental arm of the innate immune response and is best known for retrovirus restriction. Several APOBEC3 enzymes restrict HIV-1 and related retroviruses by deaminating viral cDNA cytosines to uracils compromising viral genomes. Human APOBEC3B (A3B) shows strong virus restriction activities in a variety of experimental systems, and is subjected to tight post-translational regulation evidenced by cell-specific HIV-1 restriction activity and active nuclear import. Here we ask whether lysines and/or lysine post-translational modifications are required for these A3B activities. A lysine-free derivative of human A3B was constructed and shown to be indistinguishable from the wild-type enzyme in DNA cytosine deamination, HIV-1 restriction, and nuclear localization activities. However, lysine loss did render the protein resistant to degradation by SIV Vif. Taken together, we conclude that lysine side chains and modifications thereof are unlikely to be central to A3B function or regulation in human cells.

Structural basis for targeted DNA cytosine deamination and mutagenesis by APOBEC3A and APOBEC3B.

APOBEC-catalyzed cytosine-to-uracil deamination of single-stranded (ss)DNA has beneficial functions in immunity and detrimental roles in cancer. APOBEC enzymes have intrinsic dinucleotide specificities that impart hallmark mutation signatures. Despite numerous structures, mechanisms for global ssDNA recognition and local target sequence selection remain unclear. Here, we report crystal structures of human APOBEC3A and a chimera of human APOBEC3B and APOBEC3A bound to ssDNA at 3.1 and 1.7 angstroms resolution, respectively. These structures reveal a U-shaped DNA conformation, with the specificity-conferring −1 thymine flipped out and the target cytosine inserted deep into the zinc-coordinating active site pocket. The −1 thymine base fits between flexible loops in a groove that forms upon binding ssDNA, and it makes direct hydrogen bonds with the protein accounting for the strong 5′-TC preference. These studies explain both conserved and unique properties among APOBEC family members, and provide a basis for the rational design of inhibitors to impede the evolvability of viruses and tumors.

APOBEC Enzymes: Mutagenic Fuel for Cancer Evolution and Heterogeneity.

Deep sequencing technologies are revealing the complexities of cancer evolution, casting light on mutational processes fueling tumor adaptation, immune escape, and treatment resistance. Understanding mechanisms driving cancer diversity is a critical step toward developing strategies to attenuate tumor evolution and adaptation. One emerging mechanism fueling tumor diversity and subclonal evolution is genomic DNA cytosine deamination catalyzed by APOBEC3B and at least one other APOBEC family member. Deregulation of APOBEC3 enzymes causes a general mutator phenotype that manifests as diverse and heterogeneous tumor subclones. Here, we summarize knowledge of the APOBEC DNA deaminase family in cancer, and their role as driving forces for intratumor heterogeneity and a therapeutic target to limit tumor adaptation. APOBEC mutational signatures may be enriched in tumor subclones, suggesting APOBEC cytosine deaminases fuel subclonal expansions and intratumor heterogeneity. APOBEC family members might represent a new class of drug target aimed at limiting tumor evolution, adaptation, and drug resistance.

Abstract P5-07-10: APOBEC3B drives mutagenesis in breast cancer

Abstract A typical breast cancer accumulates hundreds to thousands of somatic mutations. Breast cancer mutation spectra are often dominated by C-to-T transition mutations, which occur in a mostly dispersed manner but can also occur in localized clusters called kataegis. Several lines of evidence indicate that the innate immune DNA cytosine deaminase APOBEC3B is the primary cause of these mutations. First, APOBEC3B mRNA is up-regulated in the majority of primary breast tumors and breast cancer cell lines. Second, endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell line extracts. Third, APOBEC3B knockdown causes a corresponding decrease in genomic uracil concentrations, mutation frequencies, and C-to-T mutation levels in breast cancer cell lines. Fourth, the biochemical signature of APOBEC3B is over-represented in breast cancer mutation spectra. Fifth, breast carcinomas that express high levels of APOBEC3B have twice as many mutations as those expressing low levels. Finally, high levels of APOBEC3B associate with more aggressive disease including shorter durations of survival. All of these data are consistent with a model in which APOBEC3B-catalyzed DNA cytosine deamination provides a chronic source of DNA damage and mutagenesis in breast carcinomas that may help to explain how some tumors evolve rapidly and manifest gross heterogeneity. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-07-10.

Abstract A133: The DNA cytosine deaminase APOBEC3B drives mutagenesis in breast cancer

Abstract A typical breast cancer accumulates hundreds to thousands of somatic mutations. Breast cancer mutation spectra are often dominated by C-to-T transition mutations, which occur in a mostly dispersed manner but can also occur in localized clusters called kataegis. Several lines of evidence indicate that the innate immune DNA cytosine deaminase APOBEC3B is the primary cause of these mutations. First, APOBEC3B mRNA is up-regulated in the majority of primary breast tumors and breast cancer cell lines. Second, endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell line extracts. Third, APOBEC3B knockdown causes a corresponding decrease in genomic uracil concentrations, mutation frequencies, and C-to-T mutation levels in breast cancer cell lines. Fourth, the biochemical signature of APOBEC3B is over-represented in breast cancer mutation spectra. Fifth, breast carcinomas that express high levels of APOBEC3B have twice as many mutations as those expressing low levels. Finally, high levels of APOBEC3B associate with more aggressive disease including shorter durations of survival. All of these data are consistent with a model in which APOBEC3B-catalyzed DNA cytosine deamination provides a chronic source of DNA damage and mutagenesis in breast carcinomas that may help to explain how some tumors evolve rapidly and manifest gross heterogeneity. Citation Format: Reuben Harris. The DNA cytosine deaminase APOBEC3B drives mutagenesis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A133.

Abstract IA23: Molecular and clinical impact of APOBEC3B mutagenesis in breast cancer

Abstract Recent studies have revealed a role for the DNA cytosine deaminase APOBEC3B in breast cancer mutagenesis. First, APOBEC3B mRNA is up-regulated in the majority of primary breast tumors and breast cancer cell lines. Second, endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell line extracts. Third, APOBEC3B knockdown causes a corresponding decrease in genomic uracil concentrations, mutation frequencies, and C-to-T mutation levels in breast cancer cell lines. Fourth, the biochemical signature of APOBEC3B is evident in breast cancer mutation spectra. Fifth, breast carcinomas that express high levels of APOBEC3B have twice as many mutations as those expressing low levels. Finally, high levels of APOBEC3B associate with poorer prognostic outcomes including shorter survival durations. All of these data are consistent with a model in which APOBEC3B-catalyzed DNA cytosine deamination provides a chronic source of DNA damage and mutagenesis in breast carcinomas that helps to explain how some tumors evolve rapidly and manifest gross heterogeneity. Citation Format: Reuben S. Harris. Molecular and clinical impact of APOBEC3B mutagenesis in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA23.

Correction: Corrigendum: APOBEC3B is an enzymatic source of mutation in breast cancer

Nature 494, 366–370 (2013); doi:10.1038/nature11881 We reported a comparison of the DNA cytosine deamination context of APOBEC3B in vitro with the observed C-to-T mutation context in breast cancer (see Fig. 4c of the original Letter). We incorrectly stated in the Fig. 4c legend that the data represent all cytosines.

Cancer mutation signatures, DNA damage mechanisms, and potential clinical implications

Knowledge of cancer genomic DNA sequences has created unprecedented opportunities for mutation studies. Computational analyses have begun to decipher mutational signatures that identify underlying causes. A recent analysis encompassing 30 cancer types reported 20 distinct mutation signatures, resulting from ultraviolet light, deficiencies in DNA replication and repair, and unexpectedly large contributions from both spontaneous and APOBEC-catalyzed DNA cytosine deamination. Mutational signatures have the potential to become diagnostic, prognostic, and therapeutic biomarkers as well as factors in therapy development.

A Combined Nuclear and Nucleolar Localization Motif in Activation-Induced Cytidine Deaminase (AID) Controls Immunoglobulin Class Switching

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.

APOBEC3B and AID Have Similar Nuclear Import Mechanisms

Members of the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) protein family catalyze DNA cytosine deamination and underpin a variety of immune defenses. For instance, several family members, including APOBEC3B (A3B), elicit strong retrotransposon and retrovirus restriction activities. However, unlike the other proteins, A3B is the only family member with steady-state nuclear localization. Here, we show that A3B nuclear import is an active process requiring at least one amino acid (Val54) within an N-terminal motif analogous to the nuclear localization determinant of the antibody gene diversification enzyme AID (activation-induced cytosine deaminase). Mechanistic conservation with AID is further suggested by A3B's capacity to interact with the same subset of importin proteins. Despite these mechanistic similarities, enforced A3B expression cannot substitute for AID-dependent antibody gene diversification by class switch recombination. Regulatory differences between A3B and AID are also visible during cell cycle progression. Our studies suggest that the present-day A3B enzyme retained the nuclear import mechanism of an ancestral AID protein during the expansion of the APOBEC3 locus in primates. Our studies also highlight the likelihood that, after nuclear import, specialized mechanisms exist to guide these enzymes to their respective physiological substrates and prevent gratuitous chromosomal DNA damage.

Overlapping activation-induced cytidine deaminase hotspot motifs in Ig class-switch recombination

Ig class-switch recombination (CSR) is directed by the long and repetitive switch regions and requires activation-induced cytidine deaminase (AID). One of the conserved switch-region sequence motifs (AGCT) is a preferred site for AID-mediated DNA-cytosine deamination. By using somatic gene targeting and recombinase-mediated cassette exchange, we established a cell line-based CSR assay that allows manipulation of switch sequences at the endogenous locus. We show that AGCT is only one of a family of four WGCW motifs in the switch region that can facilitate CSR. We go on to show that it is the overlap of AID hotspots at WGCW sites on the top and bottom strands that is critical. This finding leads to a much clearer model for the difference between CSR and somatic hypermutation.

Bisulfite-mediated Deamination of Cytosine in DNA under Near-neutral Conditions

In DNA methylation analysis for epigenetic studies, the most frequently used procedure involves bisulfite-mediated conversion of cytosine to uracil. Resistance of 5-methylcytosine against this bisulfite reaction is the basis for identifying the methylated sites. This chemical modification consists of a treatment of single-stranded nucleic acids with high-concentration bisufite at pH 5-6. We report here that the treatment can be performed at pH > 6 by supplementary presence of tetramethylammonium ion. Quaternary ammonium cation appears to be essential in this unique enhancement of the reaction in the near-neutral pH range, as betaine was also effective but trimethylamine was not. Using a tetramethylammonium-bisulfite reagent at pH 6.4, we achieved a complete deamination of cytosine residues in a DNA molecule, without any significant changes taking place for 5-methylcytosine in it. These findings have opened up the possibility of improving and expanding the DNA methylation analysis technology.

Helix–hairpin–helix protein MJ1434 from Methanocaldococcus jannaschii and EndoIV homologue TTC0482 from Thermus thermophilus HB27 do not process DNA uracil residues

The mutagenic threat of hydrolytic DNA cytosine deamination is met mostly by uracil DNA glycosylases (UDG) initiating base excision repair. However, several sequenced genomes of archaeal organisms are devoid of genes coding for homologues of the otherwise ubiquitous UDG superfamily of proteins. Previously, two possible solutions to this problem were offered by (i) a report of a newly discovered family of uracil DNA glycosylases exemplified by MJ1434, a protein found in the hyperthermophilic archaeon Methanocaldococcus jannaschii, and (ii) the description of TTC0482, an EndoIV homologue from the hyperthermophilic bacterium Thermus thermophilus HB27, as being able to excise uracil from DNA. Sequence homologues of both proteins can be found throughout the archaeal domain of life. Three proteins orthologous to MJ1434 and the family founder itself were tested for but failed to exhibit DNA uracil glycosylase activity when produced in an Ung-deficient Escherichia coli host. Likewise, no DNA uracil processing activity could be detected to be associated with TTC0482, while the protein was fully active as an AP endonuclease. We propose that the uracil processing activities formerly found were due to contaminations with Ung enzyme. Use of Δung-strains as hosts for production of putatively DNA-U processing enzymes provides a simple safeguard.