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 MutS. 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.