Chapter six - AID and Somatic Hypermutation

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Abstract

In response to an assault by foreign organisms, peripheral B cells can change their antibody affinity and isotype by somatically mutating their genomic DNA. The ability of a cell to modify its DNA is exceptional in light of the potential consequences of genetic alterations to cause human disease and cancer. Thus, as expected, this mechanism of antibody diversity is tightly regulated and coordinated through one protein, activation-induced deaminase (AID). AID produces diversity by converting cytosine to uracil within the immunoglobulin loci. The deoxyuracil residue is mutagenic when paired with deoxyguanosine, since it mimics thymidine during DNA replication. Additionally, B cells can manipulate the DNA repair pathways so that deoxyuracils are not faithfully repaired. Therefore, an intricate balance exists which is regulated at multiple stages to promote mutation of immunoglobulin genes, while retaining integrity of the rest of the genome. Here we discuss and summarize the current understanding of how AID functions to cause somatic hypermutation.

Introduction

Diversity in antibodies is produced during two stages in B cell development. In pre-B cells, rearrangement of variable (V), diversity (D), and joining (J) gene segments occurs to produce the primary repertoire of immunoglobulin (Ig) receptors. In mature B cells, Ig receptors undergo affinity maturation (AM) and class switch recombination (CSR) to produce the secondary, or memory, repertoire of antibodies. The latter event occurs after antigen binds to the receptor, which initiates a dynamic cascade of cell signaling events to cause cellular activation (Gauld et al., 2002, Kurosaki, 2002, Niiro & Clark, 2002). The result of this activation is the differentiation of B cells into plasma or memory cells, which now express a large repertoire of antibodies to clear a plethora of different foreign antigens.

Diversity in the secondary repertoire is created by modifying rearranged V(D)J sequences and switching heavy chain constant genes (CH). Alteration of the V gene sequence is achieved by either direct mutagenesis or DNA strand breaks during gene conversion (GC), where strand breaks are repaired using different pseudo-V gene segments in a templated recombination mechanism. In either case, cells containing mutations that increase antibody affinity will be selected to divide and further mutate, while mutations that decrease affinity will be lost through apoptosis. Alteration of the CH gene occurs by DNA strand breaks in the switch (S) regions flanking the different CH gene exons. Breaks in two different S regions are then repaired by nonhomologous end joining to remove the intervening introns and exons. This recombination event allows of production of a defined VDJ exon with different CH gene isotypes to regulate antibody function.

A single enzyme is responsible for initiating diversity in V(D)J and CH genes: activation-induced deaminase (AID), which is a cytosine deaminase that enzymatically converts cytosine to uracil. Uracil is mutagenic when paired with guanosine in DNA, since dU mimics dT during replication, and the U:G mismatch triggers error-prone DNA repair in B cells. Thus, AID introduces somatic hypermutation (SHM) by converting dC to dU. In this chapter, the initiating events caused by AID are referred to as SHM, regardless of whether dU is found in the V or S regions. If dU occurs in V(D)J genes, SHM can produce AM or GC. If dU occurs in S regions, SHM can produce CSR. Furthermore, the proteins that process dU, such as UNG, MSH2, MSH6, and DNA polymerases, have the same activity whether dU is located in the V(D)J or S regions. Therefore, SHM, caused by AID-generated dU, underpins the three mechanisms of AM, GC, and CSR.

One key aspect of AID biology is the balance between mutagenic diversity and genomic integrity. When AID functions at non-Ig loci, both mutation and translocations can promote carcinogenesis (Ramiro et al., 2007). Thus, it is imperative to the organism that AID activity will be tightly controlled to inhibit possible oncogenic transformation, while still allowing the production of a wide diversity of antibodies. In this chapter, we highlight the intricate aspects of AID biology and regulation.

Section snippets

AID, The Master Catalyst

The mechanisms of AM, GC, and CSR were significantly advanced by the ground-breaking discovery of AID (Muramatsu et al., 1999) and its subsequent genetic analysis in humans, mice, and chickens (Arakawa et al., 2002, Rada et al., 2002b, Revy et al., 2000). Broader analysis of AID indicates that an intricate network of regulatory mechanisms controls its expression at the levels of gene transcription, mRNA stability, protein localization, protein phosphorylation, and cell signaling.

Global targeting to the Ig loci

The Ig loci are mutated in well-defined regions encoding rearranged V genes on the heavy and light chain loci, and S regions on the heavy chain locus. Sequence analysis has shown that mutation occurs in a 2-kb region around V(D)J genes (Lebecque and Gearhart, 1990) and in a 4–7-kb region around S regions (Xue et al., 2006). Thus, it can be assumed that AID functions on 10 5 to 10 6 of the genome at a given time, suggesting that precise levels of regulation target AID to such a small percentage

Deoxyuracil in DNA

Since the first identification of AID, extensive work has been performed in an attempt to elucidate the mechanism of how it promotes genomic mutation. Initial identification of the sequence similarity between AID and APOBEC1 suggested that AID may function as an RNA deaminase (Muramatsu et al., 1999). Honjo et al. (2005) proposed an RNA editing model in which AID binds to an unidentified mRNA partner in the cytoplasm and deaminates C to U. The edited mRNA would then produce a protein, perhaps

Conclusion

Even with the shear girth of information on AID biology, it is still unclear how the cell fully coordinates deamination events with mutagenic repair. As mentioned earlier, the mechanisms of AM, GC, and CSR start with a single protein, yet require extensive cellular coordination to produce the initiating deamination. It has been established that AID is tightly regulated at the levels of transcription, translation, phosphorylation, ubiquitination, cellular localization, protein stability, and

Acknowledgments

This work was supported entirely by the Intramural Research Program of the NIH, National Institute on Aging. We gratefully thank Sebastian Fugmann and Huseyin Saribasak for their insightful comments.

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