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AID deaminates C to U in either strand of Ig genes, exclusively producing C:G/G:C to T:A/A:T transition mutations if U is left unrepaired. Error-prone processing by UNG2 or mismatch repair diversifies mutation, predominantly at C:G or A:T base pairs, respectively. Here, we show that transversions at C:G base pairs occur by two distinct processing pathways that are dictated by sequence context. Within and near AGCT mutation hotspots, transversion mutation at C:G was driven by UNG2 without requirement for mismatch repair. Deaminations in AGCT were refractive both to processing by UNG2 and to high-fidelity base excision repair (BER) downstream of UNG2, regardless of mismatch repair activity. We propose that AGCT sequences resist faithful BER because they bind BER-inhibitory protein(s) and/or because hemi-deaminated AGCT motifs innately form a BER-resistant DNA structure. Distal to AGCT sequences, transversions at G were largely co-dependent on UNG2 and mismatch repair. We propose that AGCT-distal transversions are produced when apyrimidinic sites are exposed in mismatch excision patches, because completion of mismatch repair would require bypass of these sites., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Activation-induced cytidine deaminase (AID) protein initiates Ig gene mutation by deaminating cytosines, converting them into uracils. Excision of AID-induced uracils by uracil-N-glycosylase is responsible for most transversion mutations at G:C base pairs. On the other hand, processing of AID-induced G:U mismatches by mismatch repair factors is responsible for most mutation at Ig A:T base pairs. Why mismatch processing should be error prone is unknown. One theory proposes that long patch excision in G1-phase leads to dUTP-incorporation opposite adenines as a result of the higher G1-phase ratio of nuclear dUTP to dTTP. Subsequent base excision at the A:U base pairs produced could then create non-instructional templates leading to permanent mutations at A:T base pairs (1). This compelling theory has remained untested. We have developed a method to rapidly modify DNA repair pathways in mutating mouse B cells in vivo by transducing Ig knock-in splenic mouse B cells with GFP-tagged retroviruses, then adoptively transferring GFP(+) cells, along with appropriate antigen, into primed congenic hosts. We have used this method to show that dUTP-incorporation is unlikely to be the cause of AID-induced mutation of A:T base pairs, and instead propose that A:T mutations might arise as an indirect consequence of nucleotide paucity during AID-induced DNA repair.

Hypermutation of immunoglobulin genes is a key process in antibody diversification. Little is known about the mechanism, but the availability of rapid facile assays for monitoring immunoglobulin hypermutation would greatly aid the development of culture systems for hypermutating B cells as well as the screening for individuals deficient in the process. Here we describe two such assays. The first exploits the non-randomness of hypermutation. The existence of a mutational hotspot in the Ser31 codon of a transgenic immunoglobulin V gene allowed us to use PCR to detect transgene hypermutation and identify cell populations in which this mutation had occurred. For animals that do not carry immunoglobulin transgenes, we exploited the fact that hypermutation extends into the region flanking the 3'-side of the rearranged J segments. We show that PCR amplification of the 3'-flank of VDJH rearrangements that involve members of the abundantly-used VHJ558 family provides a large database of mutations where the germline counterpart is unequivocally known. This assay was particularly useful for analysing endogenous immunoglobulin gene hypermutation in several mouse strains. As a rapid assay for monitoring mutation in the JH flanking region, we show that one can exploit the fact that, following denaturation/renaturation, the PCR amplified JH flanking region DNA from germinal centre B cells yields mismatched heteroduplexes which can be quantified in a filter binding assay using the bacterial mismatch repair protein MutS -Wagner et al. (1995) Nucleic Acids Res. 23, 3944-3948-. Such assays enabled us, by example, to show that antibody hypermutation proceeds in the absence of the p53 tumour suppressor gene product.