Activation-induced cytidine deaminase (AID) initiates secondary antibody diversification processes by deaminating cytidines on single-stranded DNA. AID preferentially mutates cytidines preceded by W(A/ T)R(A/G) dinucleotides, a sequence specificity that is evolutionarily conserved from bony fish to humans. To uncover the biochemical mechanism of AID, we compared the catalytic and binding kinetics of AID on WRC (a hot-spot motif, where W equals A or T and R equals A or G) and non-WRC motifs. We show that although purified AID preferentially deaminates WRC over non-WRC motifs to the same degree observed in vivo, it exhibits similar binding affinities to either motif, indicating that its sequence specificity is not due to preferential binding of WRC motifs. AID preferentially deaminates bubble substrates of five to seven nucleotides rather than larger bubbles and preferentially binds to bubble-type rather than to single-stranded DNA substrates, suggesting that the natural targets of AID are either transcription bubbles or stem-loop structures. Importantly, AID displays remarkably high affinity for single-stranded DNA as indicated by the low dissociation constants and long half-life of complex dissociation that are typical of transcription factors and singlestranded DNA binding protein. These findings suggest that AID may persist on immunoglobulin and other target sequences after deamination, possibly acting as a scaffolding protein to recruit other factors.Activation-induced cytidine deaminase (AID) is the B-cellspecific enzyme responsible for the conversion of cytidine to uridine, the initiating event in somatic hypermutation (SHM) and class switch recombination (CSR) of antibody genes (20,22,26,29). This initial lesion can either be replicated, producing a transition mutation, or be engaged by the base excision (i.e., UNG) (34) or mismatch repair pathways (i.e., MSH2) (21, 33, 52), resulting in transversion mutations or A-T mutations, respectively. Cytidine deamination by AID also leads to the generation of double-stranded lesions in the switch regions of antibody genes, leading to CSR (9,22,42,43).While necessary for SHM and CSR, AID-induced lesions have been shown to cause chromosomal translocations contributing to lymphoma (13,36,37). Because of this inherently dangerous property of AID, it is likely that the activity of AID is controlled at different levels to focus it on immunoglobulin genes, such as through protein kinase A phosphorylation (2, 27) or the interaction with cofactors, such as replication protein A (RPA) (7). At the target DNA level, high levels of transcription have been shown to be necessary but not sufficient for AID activity (1,22,23,28,38). Since AID is able to deaminate cytidines only on single-stranded DNA (ssDNA) (6,8,10,17,18,25,47), it is likely that the requirement for transcription reflects the generation of single-stranded regions by transcription bubbles (8,22) or the generation of G4 DNA structures (11,12).Even prior to the discovery of AID, it was noted that SHM occurs more freq...

Antibody diversification necessitates targeted mutation of regions within the immunoglobulin locus by activation-induced cytidine deaminase (AID). While AID is known to act on single-stranded DNA (ssDNA), the source, structure, and distribution of these substrates in vivo remain unclear. Using the technique of in situ bisulfite treatment, we characterized these substrates—which we found to be unique to actively transcribed genes—as short ssDNA regions, that are equally distributed on both DNA strands. We found that the frequencies of these ssDNA patches act as accurate predictors of AID activity at reporter genes in hypermutating and class switching B cells as well as in Escherichia coli. Importantly, these ssDNA patches rely on transcription, and we report that transcription-induced negative supercoiling enhances both ssDNA tract formation and AID mutagenesis. In addition, RNaseH1 expression does not impact the formation of these ssDNA tracts indicating that these structures are distinct from R-loops. These data emphasize the notion that these transcription-generated ssDNA tracts are one of many in vivo substrates for AID.

Activation-induced cytidine deaminase (AID) mediates antibody diversification by deaminating deoxycytidines to deoxyuridine within immunoglobulin genes. However, it also generates genome-wide DNA lesions, leading to transformation. Though the biochemical properties of AID have been described, its 3-dimensional structure has not been determined. Hence, to investigate the relationship between the primary structure and biochemical characteristics of AID, we compared the properties of human and bony fish AID, since these are most divergent in amino acid sequence. We show that AIDs of various species have different catalytic rates that are thermosensitive and optimal at native physiological temperatures. Zebrafish AID is severalfold more catalytically robust than human AID, while catfish AID is least active. This disparity is mediated by a single amino acid difference in the C terminus. Using functional assays supported by models of AID core and surface structure, we show that this residue modulates activity by affecting ssDNA binding. Furthermore, the cold-adapted catalytic rates of fish AID result from increased ssDNA binding affinity at lower temperatures. Our work suggests that AID may generate DNA damage with variable efficiencies in different organisms, identifies residues critical in regulating AID activity, and provides insights into the evolution of the APOBEC family of enzymes.

Class switch recombination (CSR) has a fundamental function during humoral immune response and involves the induction and subsequent repair of DNA breaks in the immunoglobulin (Ig) switch regions. Here we show the role of Usp22, the SAGA complex deubiquitinase that removes ubiquitin from H2B-K120, in the repair of programmed DNA breaks in vivo. Ablation of Usp22 in primary B cells results in defects in γH2AX and impairs the classical non-homologous end joining (c-NHEJ), affecting both V(D)J recombination and CSR. Surprisingly, Usp22 depletion causes defects in CSR to various Ig isotypes, but not IgA. We further demonstrate that IgG CSR primarily relies on c-NHEJ, whereas CSR to IgA is more reliant on the alternative end joining pathway, indicating that CSR to different isotypes involves distinct DNA repair pathways. Hence, Usp22 is the first deubiquitinase reported to regulate both V(D)J recombination and CSR in vivo by facilitating c-NHEJ.

The elements which regulate gene expression have traditionally been identified by their effects on reporter genes which have been transfected into cell lines or animals. It is generally assumed that these elements have a comparable role in expression of the corresponding endogenous locus. Nevertheless, several studies of immunoglobulin heavy-chain (IgH) gene expression have reported that the requirements for expressing IgH-derived transgenes differ from the requirements for expression of the endogenous IgH locus. Thus, although expression of transgenes requires multiple elements from the J H -C intron-the E core enhancer, the matrix attachment regions (MARs) which flank E, and several switch-associated elements-B-cell lines in which expression of the endogenous heavy-chain gene is maintained at the normal level in the absence of these intronic elements have occasionally been reported. Gene targeting offers an alternative method for assessing regulatory elements, one in which the role of defined segments of endogenous genes can be evaluated in situ. We have applied this approach to the IgH locus of a hybridoma cell line, generating recombinants which bear predetermined modifications in the functional, endogenous heavy-chain gene. Our analysis indicates the following. Chromatin fibers appear to exist as loops which are anchored to the nuclear matrix, or scaffold (reviewed in reference 25). The loops are thought to correspond to functional domains in which the transcriptional state of one domain is largely independent of its neighbors. It is generally considered that the sites of anchorage in the DNA have a particular affinity for the matrix and can therefore be identified as matrix attachment regions (MARs) or scaffold attachment regions.As illustrated in Fig.