Deletions within RhoH and DNA DSBs are increased in a UNG-dependent manner in the absence of MSH2. (A) Sequence analysis of the RhoH locus identified mono- and dinucleotide deletions within [A]₁₀ (white bar) and [AG]₉ (black bar) microsatellite repeats. A total of 30 deletions were identified in 254 sequences representing ∼450 kb of sequencing data. Deletion frequency is represented as a percentage of the number of cloned sequences that contained a unique deletion. Statistical significance was calculated using Pearson’s χ² test. (B) Sequence analysis of the core IgH Sµ region identified unique intra-Sµ recombination events (white bar) and deletions (black bar) within AID hotspot motifs and repetitive sequences. A total of 25 events were identified in 228 sequences representing ∼168 kb of sequencing data. (C) Splenic B cells were obtained from wild-type mice and healthy mice deficient in UNG, MSH2, and both. Cells were activated ex vivo with lipopolysaccharide and interleukin 4 for 48 hours followed by measurement of γH2AX formation by flow cytometry. The graph depicts the mean and standard deviation of % γH2AX⁺ cells from 4 to 6 different mice of each genotype. Statistical significance was calculated by 2-tailed Student t test. (D) Representative flow cytometric analysis of γH2AX formation (y-axis) from each genotype. DAPI staining for DNA content is shown on the x-axis. Wild-type B cells treated with 20 μM etoposide for 15 minutes at the end of activation were used as a positive control for γH2AX formation. DAPI, 4,6 diamidino-2-phenylindole.