Elsevier

DNA Repair

Volume 6, Issue 12, 1 December 2007, Pages 1764-1773
DNA Repair

Alkyladenine DNA glycosylase (Aag) in somatic hypermutation and class switch recombination

https://doi.org/10.1016/j.dnarep.2007.06.012Get rights and content

Abstract

Somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes require the cytosine deaminase AID, which deaminates cytosine to uracil in Ig gene DNA. Paradoxically, proteins involved normally in error-free base excision repair and mismatch repair, seem to be co-opted to facilitate SHM and CSR, by recruiting error-prone translesion polymerases to DNA sequences containing deoxy-uracils created by AID. Major evidence supports at least one mechanism whereby the uracil glycosylase Ung removes AID-generated uracils creating abasic sites which may be used either as uninformative templates for DNA synthesis, or processed to nicks and gaps that prime error-prone DNA synthesis. We investigated the possibility that deamination at adenines also initiates SHM. Adenosine deamination would generate hypoxanthine (Hx), a substrate for the alkyladenine DNA glycosylase (Aag). Aag would generate abasic sites which then are subject to error-prone repair as above for AID-deaminated cytosine processed by Ung. If the action of an adenosine deaminase followed by Aag were responsible for significant numbers of mutations at A, we would find a preponderance of A:T > G:C transition mutations during SHM in an Aag deleted background. However, this was not observed and we found that the frequencies of SHM and CSR were not significantly altered in Aag−/− mice. Paradoxically, we found that Aag is expressed in B lymphocytes undergoing SHM and CSR and that its activity is upregulated in activated B cells. Moreover, we did find a statistically significant, albeit low increase of T:A > C:G transition mutations in Aag−/− animals, suggesting that Aag may be involved in creating the SHM A > T bias seen in wild type mice.

Introduction

Somatic hypermutation (SHM) in mammals is a process of secondary diversification of immunoglobulin (Ig) genes in activated B cells during which point mutations, and occasional insertions and deletions, are introduced into DNA encoding the antibody variable (V) regions. SHM thereby alters the affinity of antibodies for cognate antigens, a hallmark of adaptive immunity. SHM requires AID, the most likely function of which is to deaminate cytosines in the promoter proximal region DNA of Ig genes (reviewed in [1]). Deamination of cytosine (C) in DNA creates a uracil (U) base mispaired with guanine (G) that, when present in DNA, is a substrate for DNA repair. During SHM, some of the DNA repair systems that would normally faithfully repair such U:G mismatches paradoxically appear to be co-opted to generate mutations. Proteins of two major systems co-opted for SHM are the uracil glycosylase Ung, and the mismatch repair proteins Msh2, Msh6 (which comprise the Msh2/6 heterodimer), Mlh1, Pms2 (reviewed in [1], [2]) and Mlh3 [3], [4].

Mice and humans deficient for Ung are proficient for SHM, but the pattern of SHM is altered characteristically in that mutations at C and G are mainly transitions, as if uracils left unrepaired serve as a template for DNA replication [5], [6]. As a glycosylase, the function of Ung is to remove U from the DNA backbone, leaving an abasic (AP) site that is processed by concerted action of an AP endonuclease (APE1), and a deoxyribophosphodiesterase (dRPase activity of polymerase (pol) β) to produce a single-strand gap [2]. Error-free filling-in of the gap is accomplished by pol β or the high-fidelity polymerase, δ [7]. However, to explain the SHM pattern shift due to Ung-deficiency, AID/Ung-mediated single-strand gaps are likely to be filled-in using one or more translesion polymerases to generate mutations from all four nucleotides. Of the many recently identified translesion polymerases, particularly polymerase η, ι and θ have been implicated in SHM (reviewed by [8]).

Importantly, Ung-deficiency affects the SHM pattern at C (and G), but has less effect on mutations at A (and T). Mutations at A are at most 52% reduced in Ung-deficient mice [9], presumably, because Ung-dependent mutations at A can arise during long-patch base excision repair (BER) in which polymerases β or δ are replaced by translesion polymerases. In the absence of Ung, these mutations in part seem to depend on functional mismatch recognition by Msh2/6, because, although the pattern of mutations at A is not altered, their frequency is decreased from ∼50% in wildtype mice to between 26% and as little as 2% of total mutations in both Msh2−/− and Msh6−/− mice (reviewed in [1], [10]). This decrease in frequency suggests that recognition of the U:G mismatch by MutSα (the Msh2/6 heterodimer) initiates a process that ultimately leads to mutations at adenines, perhaps via recruitment of the error-prone pol η [11], [12]. Polη deficiency in mice and humans also leads to a SHM pattern characterized by a low frequency of mutations at A [12], [13], [14], [15], [16].

Currently available data suggest that mutations at A can be explained by the activities of Ung and mismatch repair, and errors created by translesion DNA polymerases. However, the precise mechanism by which these mutations arise are not yet known and possibly there are unknown factors that are involved in DNA repair during SHM that could influence mutations at A. Like mutations at C, transition mutations at A are predominant in SHM. A:T  G:C transitions could result from deamination of A to hypoxanthine (Hx), which codes as a G during DNA synthesis [17], [18]. Adenosine deaminases exist, although currently are known to act only on RNA [19]. Yet, by analogy to AID, we questioned whether a DNA adenosine deaminase might be involved in SHM. The major DNA repair enzyme for Hx is alkyladenine DNA glycosylase (Aag) [20]. In order to determine whether adenosine deamination plays a role during SHM we examined the SHM pattern in Aag−/− mice. We report here that, while the mutational pattern at A is not changed in the Aag-deficient animal, activation of B cells leads to a significant induction of Hx glycosylase activity in wild type mice. Furthermore, we see a small yet significant bias towards T:A  C:G transition mutations in the absence of Aag leading us to suggest that Aag glycosylase activity plays a role during SHM.

Section snippets

Aag−/− mice

The generation of the Aag−/− mice was previously described [20]. Aag−/− animals were backcrossed to a pure C57Bl/6J background (at least 12 backcrosses) and were 6 to 8 months old when received at the University of Chicago. Mice were analyzed soon after arrival, due to quarantine space constraints and institutional animal shipment regulations. Mice were genotyped as described [20]; genomic template DNA for PCR was derived from either PNA-low, sorted B cells from Peyer's patches, or from kidney.

Ung−/− mice

Expression of Aag in mutating and activated murine B cells

Aag is ubiquitously expressed in all tissues (NCBI Entrez Gene, GeneID: 26839), albeit at very different levels. However, in postulating a role for Aag in SHM that occurs specifically in a small subset of activated B cells during an immune response, expression of Aag was analyzed in germinal center activated B cells of immunized wildtype C57BL/6 mice. By RT-PCR, Aag is expressed in PNA-high, GL7+ (germinal center) B cells as well as other B220+ B cells, CD3+ cells (mainly T lymphocytes) and

Discussion

The findings in Aag−/− mice challenge the idea that an adenine DNA deaminase exists that operates in parallel with AID during somatic hypermutation. Aag possesses unusually broad substrate specificity for damaged DNA bases, in particular for alkylated purines, although Hx is a preferred substrate at least in vitro [32]. Moreover, Aag is reported to be the major mammalian glycosylase that functions to remove Hx from DNA in liver, testes, kidney and lung [20]. So, if deaminated A (Hx) is present

Acknowledgements

We are grateful to D. Nicolae for statistical analysis and G. Bozek for excellent technical assistance. We thank Drs. D. Barnes and T. Lindahl for Ung−/− mice. Research was funded by grants AI047380 (NIH-NIAID) and AI053130 (NIH-NIAID) to US, and P30-ES02109 (NIH/NIEHS) and 7-RO1-CA75576 (NIH/NCI) to LDS. SL was supported by an American Association of University Women International Ph.D. Fellowship. LDS is an American Cancer Society Research Professor.

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