Decrease in topoisomerase I is responsible for activation-induced cytidine deaminase (AID)-dependent somatic hypermutation

Abstract

Somatic hypermutation (SHM) and class-switch recombination (CSR) of the Ig gene require both the transcription of the locus and the expression of activation-induced cytidine deaminase (AID). During CSR, AID decreases the amount of topoisomerase I (Top1); this decrease alters the DNA structure and induces cleavage in the S region. Similarly, Top1 is involved in transcription-associated mutation at dinucleotide repeats in yeast and in triplet-repeat contraction in mammals. Here, we report that the AID-induced decrease in Top1 is critical for SHM. Top1 knockdown or haploinsufficiency enhanced SHM, whereas Top1 overexpression down-regulated it. A specific Top1 inhibitor, camptothecin, suppressed SHM, indicating that Top1's activity is required for DNA cleavage. Nonetheless, suppression of transcription abolished SHM, even in cells with Top1 knockdown, suggesting that transcription is critical. These results are consistent with a model proposed for CSR and triplet instability, in which transcription-induced non-B structure formation is enhanced by Top1 reduction and provides the target for irreversible cleavage by Top1. We speculate that the mechanism for transcription-coupled genome instability was adopted to generate immune diversity when AID evolved.DNA cleavage | non-B DNA structure T he Ig locus undergoes two types of genetic alteration-somatic hypermutation (SHM) and class switch recombination (CSR)-to generate antibody memory against given antigens when B lymphocytes are stimulated by pathogens. Both SHM and CSR depend on the expression of activation-induced cytidine deaminase (AID) (1, 2) and transcription of the target Ig loci (3-6).AID is responsible for introducing DNA cleavage in the variable (V) region and in the switch (S) region of the Ig locus for initiating SHM and CSR, respectively (7-10). AID's DNA cleavage activity is associated with its amino-terminal region because mutations at the carboxyl (C) terminus abolish CSR without affecting SHM and DNA cleavage in the S region (7,11,12). It is likely that the Cterminal region of AID is required for the recombination of the cleaved S region ends. Thus, AID carries out two separate functions with a single catalytic center located in the middle of the protein. Under normal conditions, AID is expressed only in activated B cells and introduces genetic alterations in the Ig gene. However, Ig genes are not the only targets of AID (13,14). Indeed, AID is involved in Ig-c-myc chromosomal translocation, causing malignant tumors (15, 16). More recently, certain pathogens were reported to induce the activation of AID, which may cause tumorigenesis over the long term (17)(18)(19)(20).Using an artificial construct to monitor CSR and SHM, positive correlations between transcription levels and the efficiencies of CSR and SHM have been demonstrated (21,22). Transcriptionassociated genomic instability has been extensively studied in a number of systems (23). One finding of these studies is that the frequency of mutations, including base replacements and deletions, caused...

An Evolutionary View of the Mechanism for Immune and Genome Diversity

“…To generate cleaved DNA ends, the covalently associated Top1 is probably removed by several resection enzymes, such as the MRN complex and Ctip, which are used in meiotic recombination. Consistent with this scenario, when resection of the Top1-DNA complex is blocked by the Top1 intercalating inhibitor Camptothecin (30 nM), the doublestranded break formation, SHM, and CSR are inhibited (11,12). It is well known that both the S and V regions contain direct or inverted repeats, which are prone to form non-B structures (17,41,42).…”

“…Recent findings suggest that the SHM and CSR mediated by AID have mechanisms similar to those of meiotic recombination, TAM, and triplet contraction/expansion (3)(4)(5)(11)(12)(13)(14)(15). Some of these processes are shared by V(D)J recombination (8).…”

“…First, AID activation reduces Top1 protein level; interestingly, this reduction is not seen in a loss-of-function mutant of AID (12). Second, artificial reduction of Top1 by small interfering RNA augments DNA-cleavage activity, SHM, and CSR by AID (11,12). Furthermore, in vivo, the haplo-insufficiency of Top1 (by the cross of Top1 f/+ with CAG-cre mice) dramatically augments SHM in B cells isolated from Peyer's patches (gut follicular structures in which germinal centers are continuously induced by bacterial stimulation) (11).…”

“…Second, artificial reduction of Top1 by small interfering RNA augments DNA-cleavage activity, SHM, and CSR by AID (11,12). Furthermore, in vivo, the haplo-insufficiency of Top1 (by the cross of Top1 f/+ with CAG-cre mice) dramatically augments SHM in B cells isolated from Peyer's patches (gut follicular structures in which germinal centers are continuously induced by bacterial stimulation) (11). Thus, it is clear that a reduction in Top1 correlates with enhanced DNA cleavage and a subsequent increase in SHM or CSR.…”

Accumulation of the FACT complex, as well as histone H3.3, serves as a target marker for somatic hypermutation

“…In fact, mutation targets are flanked by non-B-prone DNA sequences, such as tandem repeats and inverted repeats (3,5). It has been proposed that non-B DNA structure can cause irreversible DNA cleavage by DNA Topoisomerase I, leading to generation of the DNA lesions responsible for class switch recombination (CSR) and SHM (3,6,7).…”