Activation-induced deaminase (AID) is required for both immunoglobulin class switch recombination and somatic hypermutation. AID is known to deaminate cytidines in single-stranded DNA, but the relationship of this step to the class switch or somatic hypermutation processes is not entirely clear. We have studied the activity of a recombinant form of the mouse AID protein that was purified from a baculovirus expression system. We find that the length of the single-stranded DNA target is critical to the action of AID at the Cs positioned anywhere along the length of the DNA. The DNA sequence surrounding a given C influences AID deamination efficiency. AID preferentially deaminates Cs in the WRC motif, and additionally has a small but consistent preference for purine at the position after the WRC, thereby favoring WRCr (the lowercase r corresponds to the smaller impact on activity).

The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the S locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The S R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core S repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core S repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core S repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.Mammalian immunoglobulin (Ig) genes undergo two types of DNA recombination, in addition to a somatic hypermutation (SHM) process. V(D)J recombination occurs in early lymphocytes and assembles the variable-domain exon so that IgM can be made. Class switch recombination (CSR) occurs only at the Ig heavy-chain locus and is responsible for the change in the heavy-chain isotype from IgM to IgG, IgA, or IgE; this process is also called the heavy-chain isotype switch (6,17,29). CSR occurs at repetitive DNA elements called switch regions, which vary in sequence and length. All of the switch regions are more than 1 kb in length and consist of unit repeats of 25 to 80 bp. All are located downstream of a sterile transcript promoter, which is necessary for CSR. All have a G-rich nontemplate strand, and all are rich in sites at which a cytidine deaminase called activation-induced deaminase (AID) prefers to act, namely, WRC sites (37). The regional nature of CSR, which gave rise to the term regionally specific recombination (15), contrasts with the vast majority of other physiologic recombination systems in biology, which are regarded as site specific. The special features of switch regions (such as being long and repetitive and located downstream of a promoter, having Grich nontemplate strands, and not having sequences conserved among the different switch regions or among vertebrates that carry out CSR) suggested that the mechanism would be unusual relative to other recombination processes in biology.Investigators at the Honjo laboratory discovered the key lymphoid-specific enzyme for both CSR and SHM, AID (22,23). AID is a 26-kDa protein which deaminates C in DNA (5, 25) but only when that DNA is single stranded (2,26,36,38). A key question for CSR and SHM concerns how the DNA becomes single stranded. Because transcription appears to...

R-loops form at S␥3 and S␥2b Ig class switch regions in the chromosomes of stimulated murine primary B cells and are suspected to be a general feature of mammalian class switch regions. The in vivo upstream boundary of the R-loops is known to begin within the switch repeats. To determine how precisely the R-loop structure conforms to the repetitive zone of the murine S␥3 and S␥2b switch regions, a chemical probing method was used to obtain structural information on the downstream boundary. We find that only 61-67% of the R-loops terminate within the S␥3 and the S␥2b repetitive zones, and the remainder terminate downstream, usually within the first 600 bp immediately downstream of the core switch repeats. Interestingly, the nontemplate strand G density falls to the random level gradually through this same region. Hence, the R-loops terminate as the G-richness of the nascent RNA strand falls. This finding is consistent with thermodynamic predictions for RNA:DNA duplex strength relative to that of DNA:DNA duplexes. This result contrasts with the location of known recombination breakpoints, which correlate not with G-richness and R-loop location but rather with AGCT density. The implications of these findings are discussed in the context of models for the targeting of class switch recombination. affinity maturation ͉ immunoglobulin ͉ non-B DNA ͉ RNA:DNA hybrid O nce a mammalian B cell is producing IgM Ig, it can undergo isotype switching to produce IgG, IgA, or IgE in a process that requires a DNA recombination event called class switch recombination (CSR) (1-3). Defects in CSR result in a hyperIgM syndrome, where patients are susceptible to infections because of low concentrations of serum IgG and IgA (4). The murine IgH constant region locus has eight different constant genes (4). Each constant gene, except C␦, is 5Ј flanked by a switch region. CSR takes place between two switch regions, leading to the deletion of the intervening DNA segment (5, 6). Mammalian switch regions vary substantially in primary sequence, but all have the following features: highly repetitive, G-rich on the nontemplate strand, and 1-12 kb in length. The switch repeats contain two short subrepeats: AGCT and GGGGT predominate in S, S␣, and S. S␥1, S␥2a, S␥2b, and S␥3 include numerous AGCT sites and clusters of G's also (7). Most CSR breakpoints are identified in the switch regions; however, they are less frequently found within the several hundred base pairs upstream or downstream (7,8).Germ-line transcription is an important feature of CSR (1, 9). Upon antigen stimulation, cytokines stimulate the appropriate promoter in front of switch regions. The unspliced germ-line transcript consists of the noncoding I exon, S region, and the corresponding C exon. It does not encode protein and, hence, is called the sterile transcript. Without transcription, CSR is markedly reduced (10-12).Several DNA structures have been proposed to form in the switch region. By computer modeling, a DNA stem-loop structure was suggested in Xenopus and mammalian switch ...

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