Binding of AID to DNA Does Not Correlate with Mutator Activity

Abstract

The DNA deaminase AID initiates somatic hypermutation (SHM) and class switch recombination (CSR) by deaminating cytidines to uridines at variable region (V) genes and switch (S) regions. The mechanism by which AID is recruited to V genes and S region DNA is poorly understood. Here we have employed the CH12 B lymphoma line to demonstrate that while S regions can efficiently recruit AID and undergo mutations and deletions, AID neither binds to nor mutates the V gene, thus clearly demonstrating intra-immunoglobulin locus specificity. Depletion of the RNA-binding protein Ptpb2, previously shown to promote recruitment of AID to S regions, enables stable association of AID with the V gene. Surprisingly, AID binding to the V gene does not induce SHM. These results unmask a striking lack of correlation between AID binding and its mutator activity, providing evidence for the presence of factors required downstream of AID binding to effect SHM. Furthermore, our findings suggest that S regions are preferred targets for AID and, aided by Ptbp2, act as “sinks” to sequester AID activity from other genomic regions.

DNA polymerase β prevents AID-instigated mutagenic non-canonical mismatch DNA repair

“…The overall mutation frequency did not differ significantly between wild type parental (WT) and Polb knockdown CH12-F3 cells (2.1 x10 -3 vs 2.5 x10 -3 , respectively, Table I). We found that mutations at A:T base pairs were infrequent in the WT CH12-F3 cells ( 9 out of 59363 sequenced A:T bps were mutated), in agreement with a previous study (Matthews et al, 2014). In contrast, Polb knockdown CH12-F3 cells showed a considerable number of A:T mutations (23 out of 57988 sequenced A:T bps were mutated.…”

“…In addition, these cells accrue AID-dependent point mutations in the Ig switch μ region (Sμ) upon stimulation, similar to mouse splenic B cells Schrader et al, 2003). Strikingly, despite a high frequency of AID-dependent mutations 5' of the Sμ repetitive region, the CH12-F3 cells conspicuously lack A:T mutations for hitherto unknown reasons (Matthews et al, 2014). We hypothesized that the late gap-filling and repair activities of Polβ and LigIII diminishes entry points for Exo1, thereby suppressing A:T mutagenesis.…”

“…Of interest, in vitro systems to study AID-instigated mutagenesis are characterized by the low frequency of A:T mutations versus in vivo B cells (approximately 0-20% versus 50-60% of all mutations, respectively) (Martin et al, 2002;Matthews et al, 2014;Xiao et al, 2007;Yoshikawa et al, 2002). We hypothesized that this apparent discrepancy is caused by distinct DNA repair activities in vitro versus in vivo.…”

AID Recognizes Structured DNA for Class Switch Recombination

“…In comparison, SHM can be induced by CSR-deficient AID variants in cells (Hwang et al, 2015; Phamet al, 2016) and by APOBEC3 homologs during retroviral infection (Halemano et al, 2014), suggesting that mutations in Ig V regions may not require all the AID features employed in CSR. Because chromatin immunoprecipitation sequencing data showed that Ig V regions may not bind AID as stably as S regions (Matthews et al, 2014b), and the assistant patch mutant R174S also causes SHM deficiency (Durandy et al, 2006; Honjo et al, 2012), we speculate that Ig V regions may only transiently recruit AID using local secondary structures like branched DNA, which do not induce AID oligomerization and stable association.…”

IgH Chain Class Switch Recombination: Mechanism and Regulation

“…The splicing regulator PTBP2, a protein that binds AID and S region transcripts, is important for efficient CSR. Knockdown of this protein reduces the association of AID to S regions (51, 52). PTBP2 is known to regulate alternative splicing and many aspects of RNA metabolism (53), although its specific role(s) in CSR is not defined.…”