Uracil-DNA Glycosylase in Base Excision Repair and Adaptive Immunity

Doseth, Berit; Visnes, Torkild; Wallenius, Anders; Ericsson, Ida; Sarno, Antonio; Pettersen, Henrik Sahlin; Flatberg, Arnar; Catterall, Tara; Slupphaug, Geir; Krokan, Hans E.; Kavli, Bodil

doi:10.1074/jbc.m111.230052

Cited by 47 publications

(44 citation statements)

References 36 publications

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“…S3 in the supplemental material). Thus, despite recent reports that SMUG1 contributes more to uracil excision than UNG2 in mice (36) and that SMUG1 contributes to CSR and SHM in mice (38), we found that UNG2 is the only uracilspecific DNA glycosylase whose expression is upregulated in response to B cell stimulation. Thus, it must be responsible for the repair of an overwhelming majority of uracils created by AID in stimulated WT B lymphocytes.…”

Section: Resultscontrasting

confidence: 51%

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Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Shalhout

Haddad

Sosin

et al. 2014

Molecular and Cellular Biology

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d Activation-induced deaminase (AID) converts DNA cytosines to uracils in immunoglobulin genes, creating antibody diversification. It also causes mutations and translocations that promote cancer. We examined the interplay between uracil creation by AID and its removal by UNG2 glycosylase in splenocytes undergoing maturation and in B cell cancers. The genomic uracil levels remain unchanged in normal stimulated B cells, demonstrating a balance between uracil generation and removal. In stimulated UNG ؊/؊ cells, uracil levels increase by 11-to 60-fold during the first 3 days. In wild-type B cells, UNG2 gene expression and enzymatic activity rise and fall with AID levels, suggesting that UNG2 expression is coordinated with uracil creation by AID. Remarkably, a murine lymphoma cell line, several human B cell cancer lines, and human B cell tumors expressing AID at high levels have genomic uracils comparable to those seen with stimulated UNG ؊/؊ splenocytes. However, cancer cells express UNG2 gene at levels similar to or higher than those seen with peripheral B cells and have nuclear uracil excision activity comparable to that seen with stimulated wild-type B cells. We propose that more uracils are created during B cell cancer development than are removed from the genome but that the uracil creation/excision balance is restored during establishment of cell lines, fixing the genomic uracil load at high levels. When B lymphocytes are activated through antigen presentation, they acquire hypermutations in the immunoglobulin (Ig) genes, facilitating affinity maturation of antibodies. An enzyme, activation-induced deaminase (AID), initiates these events by converting cytosines in DNA to uracil (1-4). The introduction of this rare base into DNA leads to a very high frequency of base substitution mutations in the Ig variable domain (known as somatic hypermutations [SHMs]; reviewed in references 5 and 6). Generation of uracils is also the first step in the creation of strand breaks in the switch regions of Ig genes, leading to the replacement of the constant domain with other domains such as ␥, in a process called class-switch recombination (CSR; reviewed in reference 7). AID also binds near the transcription start sites of a large number of actively transcribed genes (8) and mutates a number of additional genes, including those encoding BCL-6, MYC, PAX-5, and PIM1 (9-12). The uracils generated by AID are thought to be removed by the nuclear form of the uracil-DNA glycosylase, UNG2, creating abasic sites that are repaired by error-prone copying mechanisms causing hypermutations (13,14). Another uracil-DNA glycosylase, SMUG1, is normally considered the backup enzyme for UNG2 (15), but overproduction of SMUG1 is required for it to complement an UNG Ϫ/Ϫ mutant during CSR (16). Additionally, DNA mismatch repair (MMR) also plays a role in shaping the mutation spectrum in SHM (17).There is a strong connection between expression of AID and cancers in animals. Constitutive expression of AID in mice causes T cell cancers (18). Many human...

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Section: Resultscontrasting

confidence: 51%

“…The principal function of UNG2 in cells is to excise uracils introduced into DNA during replication through dUTP utilization by replicative polymerases (15,36). Consequently, UNG2 protein in B cells may have to be recruited to loci where AID acts.…”

Section: Discussionmentioning

confidence: 99%

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Shalhout

Haddad

Sosin

et al. 2014

Molecular and Cellular Biology

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“…Finally, the difference that emerges from a comparison between the data from mouse and humans could reflect a species difference in the biochemical activity of SMUG1 and/or the operation of the MSH2‐initiated pathway. However, it could be also very well that the few UNG‐deficient patients analyzed so far represent a clinical ascertainment bias as they were identified on the basis of their immunological phenotype.…”

Section: Discussionmentioning

confidence: 99%

Uracil excision by endogenous SMUG1 glycosylase promotes efficient Ig class switching and impacts on A:T substitutions during somatic mutation

et al. 2014

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Excision of uracil introduced into the immunoglobulin loci by AID is central to antibody diversification. While predominantly carried out by the UNG uracil‐DNA glycosylase as reflected by deficiency in immunoglobulin class switching in Ung−/− mice, the deficiency is incomplete, as evidenced by the emergence of switched IgG in the serum of Ung−/− mice. Lack of switching in mice deficient in both UNG and MSH2 suggested that mismatch repair initiated a backup pathway. We now show that most of the residual class switching in Ung−/− mice depends upon the endogenous SMUG1 uracil‐DNA glycosylase, with in vitro switching to IgG1 as well as serum IgG3, IgG2b, and IgA greatly diminished in Ung−/−Smug1−/− mice, and that Smug1 partially compensates for Ung deficiency over time. Nonetheless, using a highly MSH2‐dependent mechanism, Ung−/−Smug1−/− mice can still produce detectable levels of switched isotypes, especially IgG1. While not affecting the pattern of base substitutions, SMUG1 deficiency in an Ung−/− background further reduces somatic hypermutation at A:T base pairs. Our data reveal an essential requirement for uracil excision in class switching and in facilitating noncanonical mismatch repair for the A:T phase of hypermutation presumably by creating nicks near the U:G lesion recognized by MSH2.

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“…Alternatively, there could be a difference in the mechanism between the species (19). For instance, the uracil DNA glycosylase SMUG1 is more abundant in mice than humans (45), and could partly compensate for UNG in producing serum Ig. However, given SMUG1's inability to potentiate CSR in Ung 2/2 Msh2 2/2 or Ung 2/2 Msh6 2/2 mice (10, 11), this seems unlikely.…”

Section: Discussionmentioning

confidence: 99%

Separation of Function between Isotype Switching and Affinity Maturation In Vivo during Acute Immune Responses and Circulating Autoantibodies in UNG-Deficient Mice

Zahn

Daugan

Safavi

et al. 2013

The Journal of Immunology

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Activation-induced deaminase converts deoxycytidine to deoxyuridine at the Ig loci. Complementary pathways, initiated by the uracil-DNA glycosylase (UNG) or the mismatch repair factor MSH2/MSH6, must process the deoxyuridine to initiate class-switch recombination (CSR) and somatic hypermutation. UNG deficiency most severely reduces CSR efficiency and only modestly affects the somatic hypermutation spectrum in vitro. This would predict isotype-switching deficiency but normal affinity maturation in Ung−/− mice in vivo, but this has not been tested. Moreover, puzzling differences in the amount of circulating Ig between UNG-deficient humans and mice make it unclear to what extent MSH2/MSH6 can complement for UNG in vivo. We find that Ab affinity maturation is indeed unaffected in Ung−/− mice, even allowing IgM responses with higher than normal affinity. Ung−/− mice display normal to only moderately reduced basal levels of most circulating Ig subclasses and gut-associated IgA, which are elicited in response to chronically available environmental Ag. In contrast, their ability to produce switched Ig in response to immunization or vesicular stomatitis virus infection is strongly impaired. Our results uncover a specific need for UNG in CSR for timely and efficient acute Ab responses in vivo. Furthermore, Ung−/− mice provide a novel model for separating isotype switching and affinity maturation during acute (but not chronic) Ab responses, which could be useful for dissecting their relative contribution to some infections. Interestingly, Ung−/− mice present with circulating autoantibodies, suggesting that UNG may impinge on tolerance.

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Uracil-DNA Glycosylase in Base Excision Repair and Adaptive Immunity

Cited by 47 publications

References 36 publications

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Genomic Uracil Homeostasis during Normal B Cell Maturation and Loss of This Balance during B Cell Cancer Development

Uracil excision by endogenous SMUG1 glycosylase promotes efficient Ig class switching and impacts on A:T substitutions during somatic mutation

Separation of Function between Isotype Switching and Affinity Maturation In Vivo during Acute Immune Responses and Circulating Autoantibodies in UNG-Deficient Mice

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