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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Activation-induced cytidine deaminase (AID) is a 'master molecule' in immunoglobulin (Ig) class-switch recombination (CSR) and somatic hypermutation (SHM) generation, AID deficiencies are associated with hyper-IgM phenotypes in humans and mice. We show here that recessive mutations of the gene encoding uracil-DNA glycosylase (UNG) are associated with profound impairment in CSR at a DNA precleavage step and with a partial disturbance of the SHM pattern in three patients with hyper-IgM syndrome. Together with the finding that nuclear UNG expression was induced in activated B cells, these data support a model of CSR and SHM in which AID deaminates cytosine into uracil in targeted DNA (immunoglobulin switch or variable regions), followed by uracil removal by UNG. [ABSTRACT FROM AUTHOR]

Mice deficient in the Ung uracil-DNA glycosylase have an increased level of uracil in their genome, consistent with a major role of Ung counteracting U?:?A base pairs arising by misincorporation of dUMP during DNA replication. A complementary uracil-excising activity apparently acts on premutagenic U?:?G lesions resulting from deamination of cytosine throughout the genome. However, Ung specifically processes U?:?G lesions targeted to immunoglobulin variable (V) genes during somatic hypermutation and class-switch recombination. Gene-targeted Ung-/- null mice remained tumour-free and showed no overt pathological phenotype up to ~12 months of age. We have monitored a large cohort of ageing Ung-/- mice and, beyond 18 months of age, they had a higher morbidity than Ung+/+ controls. Post-mortem analyses revealed pathological changes in lymphoid organs, abnormal lymphoproliferation, and a greatly increased incidence of B-cell lymphomas in older Ung-deficient mice. These are the first data reporting the development of spontaneous malignancies in mice due to deficiency in a DNA glycosylase. Furthermore, they support a specific role for Ung in the immune system, with lymphomagenesis being related to perturbed processing of antibody genes in germinal centre B cells.Oncogene (2003) 22, 5381-5386. doi:10.1038/sj.onc.1206860 [ABSTRACT FROM AUTHOR]

Highlights: [•] E2A binds to the UNG2 promoter and represses expression of UNG2. [•] UNG2 preferentially accumulates in regions of the IgH gene containing AID hotspots. [•] Data indicate that this UNG2 accumulation is negatively regulated by E2A as well. [•] Over-expression of E2A can suppress class switch recombination. [•] The results suggest that E2A is regulating the balance between AID and UNG2. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: In human cell nuclei, UNG2 is the major uracil-DNA glycosylase initiating DNA base excision repair of uracil. In activated B cells it has an additional role in facilitating mutagenic processing of AID-induced uracil at Ig loci and UNG-deficient patients develop hyper-IgM syndrome characterized by impaired class-switch recombination and disturbed somatic hypermutation. How UNG2 is recruited to either error-free or mutagenic uracil processing remains obscure, but likely involves regulated interactions with other proteins. The UNG2 N-terminal domain contains binding motifs for both proliferating cell nuclear antigen (PCNA) and replication protein A (RPA), but the relative contribution of these interactions to genomic uracil processing is not understood. Interestingly, a heterozygous germline single-nucleotide variant leading to Arg88Cys (R88C) substitution in the RPA-interaction motif of UNG2 has been observed in humans, but with unknown functional relevance. Here we demonstrate that UNG2-R88C protein is expressed from the variant allele in a lymphoblastoid cell line derived from a heterozygous germ line carrier. Enzyme activity as well as localization in replication foci of UNG2-R88C was similar to that of WT. However, binding to RPA was essentially abolished by the R88C substitution, whereas binding to PCNA was unaffected. Moreover, we show that disruption of the PCNA-binding motif impaired recruitment of UNG2 to S-phase replication foci, demonstrating that PCNA is a major factor for recruitment of UNG2 to unperturbed replication forks. Conversely, in cells treated with hydroxyurea, RPA mediated recruitment of UNG2 to stalled replication forks independently of functional PCNA binding. Modulation of PCNA- versus RPA-binding may thus constitute a functional switch for UNG2 in cells subsequent to genotoxic stress and potentially also during the processing of uracil at the immunoglobulin locus in antigen-stimulated B cells. [Copyright &y& Elsevier]

Genomic uracil is a DNA lesion but also an essential key intermediate in adaptive immunity. In B cells, activation-induced cytidine deaminase deaminates cytosine to uracil (U:G mispairs) in Ig genes to initiate antibody maturation. Uracil-DNA glycosylases (UDGs) such as uracil N-glycosylase (UNG), single strand-selective monofunctional uracil-DNA glycosylase 1 (SMUG1), and thymine-DNA glycosylase remove uracil from DNA. Gene-targeted mouse models are extensively used to investigate the role of these enzymes in DNA repair and Ig diversification. However, possible species differences in uracil processing in humans and mice are yet not established. To address this, we analyzed UDG activities and quantities in human and mouse cell lines and in splenic B cells from Ung+/+ and Ung-/- backcrossed mice. Interestingly, human cells displayed 15- fold higher total uracil excision capacity due to higher levels of UNG. In contrast, SMUG 1 activity was -8-fold higher in mouse cells, constituting -50% of the total U:G excision activity com- pared with less than 1% in human cells. Inactivated B cells, both UNG and SMUG 1 activities were at levels comparable with those measured for mouse cell lines. Moreover, SMUG 1 activity per cell was not down-regulated after activation. We therefore suggest that SMUG 1 may work as a weak backup activity for UNG2 during class switch recombination in Ung~ mice. Our results reveal significant species differences in genomic uracil processing. These findings should be taken into account when mouse models are used in studies of uracil DNA repair and adaptive immunity. [ABSTRACT FROM AUTHOR]