Fig 2: HMCES deficiency leads to increased deletions in SHM target regions. (A) Representative flow cytometry histogram plot of and graph quantitating GFP loss in RASH-1C cells treated with empty vector or a HMCES sgRNA without or with 4-d dox treatment. (B) Western blot showing HMCES protein levels in RASH-1C cells treated with two different HMCES sgRNAs. (C) Western blot showing HMCES protein levels in 11 independent HMCES KO RASH-1C cell clones. (D) Dot plot of GFP loss in cells shown in C treated with dox for 4 d. (E) Scatter plot of GFP loss versus IgM loss from WT and HMCES KO RASH-1C cell clones treated with dox for 4 d. (F) Western blot showing HMCES protein levels in six independent HMCES KO Ramos cell clones. (G) IgM loss from WT, HMCES KO, or AID KO Ramos cell clones at different time points of culture. (H,I) Deletion frequency in the HTS7 (H) and VDJ (I) regions from WT, HMCES KO, UNG KO, and HMCES/UNG dKO RASH-1C cells with dox for 4 d. Deletion frequency was calculated as (number of deletions/number of sequences) × 100; fold changes compared with WT are marked above the bar. (J,K) Distribution of deletion lengths in HTS7 (J) and VDJ (K) regions from WT, HMCES KO, UNG KO, and HMCES/UNG dKO RASH-1C cells treated with dox for 4 d. Deletions were divided into three groups based on length as indicated and the percentage of sequences with indicated deletion length are shown. Fold changes compared with WT are marked above the bar. (L,M) Deletion frequency in the IGH VDJ (L) and IGL VJ (M) regions from WT, HMCES KO, and AID KO Ramos cells. Single cells were seeded and grown for 6 wk and then harvested for analysis. Fold changes compared with WT are marked above the bar. (N,O) Distribution of deletion lengths in the IGH VDJ (N) and IGL VJ (O) regions from WT, HMCES KO, and AID KO Ramos cells. Deletions were divided into three groups based on length as indicated and the percentage of sequences with the indicated deletion length are shown. Fold changes compared with WT are marked above the bar. (P) Deletion profile in the VDJ region from WT and HMCES KO RASH-1C cells treated with dox for 4 d. AID hotspots are indicated with a vertical line at the X-axis. The CDR1, CDR2, and CDR3 regions are shaded. (Q) Deletion profile in the VDJ region from WT, HMCES KO, and AID KO Ramos cells. AID hotspots are indicated with a vertical line at the X-axis. The CDR1, CDR2, and CDR3 regions are shaded. Throughout the figure, data are presented with the bars representing mean and the error bars as ±SD. Statistical significance was calculated using two-tailed Student's t-test for A and D and using one way ANOVA with Dunnett's post-test for G–O. (***) P-value < 0.001, (**) P-value < 0.01, (*) P-value < 0.05, (ns) not significant.

Fig 3: HMCES deficiency leads to increased deletions in mouse germinal center B cells. (A,B) Deletion frequencies in the region downstream from the Igh J4 gene segment (JH4 intron) in germinal center B (GCB) cells from either the spleen (A) or Peyer's patch (B) of Hmces-/- and control Hmces+/- and Hmces+/+ mice. (C,D) Distribution of deletion lengths in the JH4 intron in splenic (C) and Peyer's patch GCB (D) cells from Hmces-/- and control Hmces+/+ and Hmces+/- mice. (E,F) Insertion frequencies in the JH4 intron in splenic (E) and Peyer's patch GCB (F) cells from Hmces-/- and control Hmces+/+ and Hmces+/- mice. (G,H) Point mutation frequencies in the JH4 intron in splenic (G) and Peyer's patch GCB (H) cells from Hmces-/- and control Hmces+/+ and Hmces+/- mice. (I,J) Mutation frequencies for G/C transitions, G/C transversions, and A/T mutations in the JH4 intron in splenic (I) or Peyer's patch GCB (J) cells from Hmces-/- and control Hmces+/+ and Hmces+/- mice. (K) Flow cytometry plots of GCB cells gated as the FAS+ CD38- population among total B cells in spleens of Hmces+/+ and Hmces-/- mice 14 d after immunization with NP-CGG. (L,M) Bar graphs showing the frequencies of GCB cells in Hmces+/+ or Hmces+/- and Hmces-/- mice in spleens (L) or Peyer's patches (M). (N) Bar graphs quantifying the levels of low- and high-affinity IgM antibodies measured by ELISA in the sera of Hmces-/- and control Hmces+/+ and Hmces+/- mice. NP-BSA with a conjugation ratio of 27 and 2 was used to detect low-and high-affinity antibodies, respectively. (O) Working model for the mechanism by which HMCES suppresses deletions during SHM. After AID acts, MSH2/6, EXO1, and likely other factors can resect one strand to expose a region of ssDNA containing a U residue, on which UNG can act to create an abasic site (AP site). Cleavage of this vulnerable intermediate by APE2 would result in a DNA double-strand break (DSB), which would be prone to end resection by exonucleases (including APE2 itself) and to generating a deletion. HMCES protects against this outcome by forming a covalent cross-link with the abasic site, thereby blocking cleavage by APE2. The events that occur subsequent to HMCES cross-linking are not known but could involve translesion synthesis (TLS) by an error-prone polymerase. The pathway depicted here is just one of the pathways depicted in Figure 1A (the second from the top). Throughout the figure, data are presented with the bars representing mean and the error bars as ±SD. Statistical significance was calculated using two-tailed Student's t-test. (***) |P-value < 0.001, (**) P-value < 0.01, (ns) not significant.

Fig 4: HMCES-Deficient Cells Are Hypersensitive to Nuclear APOBEC3A Expression, Which Promotes HMCES Localization to the Chromatin(A) Representative images of a colony-viability assay in hTERT-RPE-1 GFP-APOBEC3A-ERT2 cells transfected with non-targeting control (NT) or HMCES (H-1 and H-2) siRNAs and treated with 4-OHT for 24 h.(B and C) Quantitation of clonogenic survival assays for RPE A3A (B) and HCT116 A3A (C). Means and SD of 3 experiments. p values were derived from an ANOVA with Holm-Sidak post-test.(D and E) Quantitation of the insoluble HMCES PLA signal after 4-OHT treatment in EdU-positive (D) and EdU-negative (E) hTERT-RPE-1 cells, with and without stable expression of GFP-APOBEC3A-ERT2. (-1°, no primary HMCES antibody). Each data point represents the HMCES-PLA-integrated nuclear intensity in one cell. Bars represent the median, and p values were derived from an ANOVA with Dunn’s multiple comparisons post-test. See also Figures S1 and S2.

Fig 5: The function of HMCES in SHM is dependent on abasic site cross-linking and ssDNA binding residues. (A) Schematic of human HMCES protein. (B) Western blot of HMCES and AID in WT or HMCES KO RASH-1C cells reconstituted with WT HMCES or its mutants and induced with dox for 4 d. (C,D) WT HMCES or the indicated HMCES mutants were expressed in RASH-1C cells in which HMCES was knocked out (RASH-1C HMCES KO) or intact (RASH-1C WT). Cells induced with dox for 4 d were assayed for GFP loss. (E) WT HMCES or the indicated HMCES mutants were expressed in HMCES KO RASH-1C cells. Cells induced with dox for 4 d were assayed for IgM loss. (F,G) Deletion frequencies in HTS7 (F) and VDJ (G) from HMCES KO RASH-1C cells reconstituted with WT HMCES or its mutants or overexpressing UNG2, as indicated. (H,I) Distribution of deletion lengths in the HTS7 (H) and VDJ (I) regions from HMCES KO RASH-1C cells reconstituted with WT HMCES or its mutants or overexpressing UNG2. (J,K) Point mutation frequencies in the HTS7 (J) and VDJ (K) regions from HMCES KO RASH-1C cells reconstituted with WT HMCES or its mutants or overexpressing UNG2. Throughout the figure, data are presented with the bars representing mean and the error bars as ±SD. Statistical significance was calculated using one-way ANOVA with Dunnett's post-test. (***) P-value < 0.001, (**) P-value < 0.01, (*) P-value < 0.05, (ns) not significant.