The Function of AID in Somatic Mutation and Class Switch Recombination

Abstract

The immune system has evolved specific mechanisms to combat a potentially limitless number of foreign pathogens using a limited arsenal of Ig genes. To diversify the coding potential of the Ig genes, B cells undergo several processes of regulated genetic alterations. Early in their development, B cells in the bone marrow undergo V(D)J recombination to juxtapose variable region V, D, and J segments in different combinations, creating a large repertoire of antibodies (1). Later in B cell development, usually after antigen-dependent activation of B cells, the genetic alteration processes of somatic mutation (SM), class switch recombina-tion (CSR), and gene conversion further diversify the antigen-recognition repertoire as well as the effector function of encoded antibodies. In SM, which is the dominant means of secondary alteration of variable region gene sequences in humans and mice, mutations are introduced in the Ig variable region genes at a tremendous rate, which allows for evolution of high affinity antibodies (2). In some vertebrates, such as chickens and pigs, diversification of assembled Ig variable regions occurs by a gene conversion mechanism rather than SM (3). In CSR, to diversify the effector function of specific antibodies, recombination occurs within the downstream portion of the IgH locus to join variable region genes with different constant (C H) region genes (4). SM introduces mutations, small deletions, and insertions at a high rate in a 2 kb region downstream of the Ig promoter , altering the specificities of the encoded antibodies (2). SM usually occurs within the specific microenviron-ment of germinal centers, which is thought to be critical for this process. Within germinal centers, antibodies with high affinity for antigen are then selected, while low-affinity antibodies are weeded out in a process termed affinity maturation. The SM mutations commonly occur at conserved sequence motifs (hotspots). The mechanism of SM has been proposed to involve generation of DNA breaks followed by a repair process that involves an error-prone polymerase (5). In gene conversion, the assembled variable region sequences are altered via homologous recombina-tion using other unrearranged variable region genes or pseudogenes as templates. DNA breaks that occur during SM were first detected by overexpressing the enzyme terminal deoxynucleotidyl transferase (TdT), which catalyzes nontemplated addition of nucleotides to free DNA ends, in a constitutively hyper-mutating B cell line (6). This study revealed that nucleotides were specifically inserted at SM hotspots, suggesting that these hotspots were sites of DNA breaks. Subsequently , three groups investigated the nature of these breaks (single versus double-strand breaks, blunt versus staggered or hairpin ends) using ligation-mediated PCR (LM-PCR) strategies. These studies detected blunt-ended double-stranded DNA breaks (DSBs) preferentially at hot-spot sequences in B cells undergoing SM (7, 8). Papavasil-iou and Schatz further showed that the majority of the DSBs were detected during the G2 cell cycle phase when the homologous recombination repair pathway is dominant (7), while Bross et al. demonstrated the reliance of these breaks on transcriptional activity (8). In addition, Kong and Maizels also detected single-stranded DNA breaks at hy-permutation sites (9). Although breaks in genomic DNA can arise by a number of mechanisms, including apoptotic DNA fragmentation and in vitro shearing, these studies provided considerable evidence that the breaks detected by the LM-PCR assays were associated with the SM mechanism. Specifically, they showed that the breaks occurred preferentially at SM hotspots, were dependent on transcription , and occurred preferentially in hypermutating B cells. CSR is another genetic modification employed by B cells to boost the immune response by changing the constant region of the antibody while retaining the antigen-specific variable region. This DNA recombination event occurs between two switch (S) regions, consisting of stretches of repetitive sequence, located just upstream of each C H gene (except C). CSR is a recombination/dele-tion mechanism that juxtaposes a downstream C H (C , C , or C) to the expressed V(D)J segment, allowing switching from expression of IgM to IgG, IgA, or IgE (4). In vivo, CSR requires germline transcription of S region sequences, the generation of DSBs within S regions, and resolution of these breaks by a process that requires NHEJ factors (4). It has been suggested that CSR may, like SM, involve DNA