Ectopic restriction of DNA repair reveals that UNG2 excises AID-induced uracils predominantly or exclusively during G1 phase.

Immunoglobulin (Ig) affinity maturation requires the enzyme AID, which converts cytosines (C) in Ig genes into uracils (U). This alone produces C:G to T:A transition mutations. Processing of U:G base pairs via U N-glycosylase 2 (UNG2) or MutSα generates further point mutations, predominantly at G:C or A:T base pairs, respectively, but it is unclear why processing is mutagenic. We aimed to test whether the cell cycle phase of U processing determines fidelity. Accordingly, we ectopically restricted UNG2 activity in vivo to predefined cell cycle phases by fusing a UNG2 inhibitor peptide to cell cycle-regulated degradation motifs. We found that excision of AID-induced U by UNG2 occurs predominantly during G1 phase, inducing faithful repair, mutagenic processing, and class switching. Surprisingly, UNG2 does not appear to process U:G base pairs at all in Ig genes outside G1 phase.

Incorporation of dUTP does not mediate mutation of A:T base pairs in Ig genes in vivo.

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.