Fig 2: NRH treatment increased cellular NAD+ levels and promoted PARP1 activity upon TMZ treatment. (A) Total cellular NAD+ levels in LN428/SCR-gRNA or LN428/MSH6-KO cells after treatment with NRH (100 μM) for the time periods indicated; (** p < 0.01, one-way ANOVA; NS = not significant). (B) PAR (poly-ADP-ribose) immunoblot analysis of total cell lysates (LN428/SCR cells, top panel; LN428/MSH6-KD, bottom panel) after treatment with NRH (100 μM) for 4 h and then TMZ at the indicated doses for an additional 1 h, as compared to cells treated with DMSO. β-Actin was used as the loading control. (C) PAR (poly-ADP-ribose) immunoblot analysis of total cell lysates (LN428/SCR cells, left panel; LN428/MSH6-KD, right panel) after treatment with PARGi (100 μM) or N + P for 4 h and then TMZ at the indicated doses for an additional 1 h, as compared to cells treated with PARGi. β-Actin was used as the loading control.

Fig 3: PAR accumulation suppresses cell survival signaling and induces apoptosis. (A) Immunoblot analysis of total cell lysates for PAR, γ-H2AX, and cleaved caspase 3 at 24 h after treatment of LN428/MSH6-KD cells with DMSO, NRH (100 μM), PARGi (10 μM), N + P, TMZ (250 μM), T + N, T + P, or T + N + P. β-Actin was used as the loading control. (B) Relative caspase 3/7 activity (LN428/MSH6-KD cells) 24 h after treatment with DMSO, NRH (100 μM), PARGi (10 μM), N + P, TMZ (250 μM), T + N, T + P, or T + N + P; normalized to DMSO (** p < 0.01, **** p < 0.0001, one-way ANOVA). (C) Total cellular NAD+ levels in LN428/MSH6-KD cells treated with DMSO, NRH (100 μM), PARGi (10 μM), N + P, TMZ (250 μM), T + N, T + P, or T + N + P for 24 h, normalized to DMSO (** p < 0.01, **** p < 0.0001, one-way ANOVA). (D) Immunoblot analysis of total cell lysates for MSH6, PAR, p-AKT, γ-H2AX, and cleaved caspase 3, 24 h after treatment of MSH6-WT or MSH6-KO from LN428, T98G, or U87MG cells treated with DMSO, TMZ (125 μM), T + P, or T + N + P. β-Actin was used as the loading control.

Fig 4: Mismatch repair factors contribute to deletions caused by HMCES deficiency. (A–C) MSH2, MSH6, or EXO1 was knocked out in WT or HMCES KO RASH-1C cells. Western blots of MSH2 (A), MSH6 (B), and EXO1(C) in WT or HMCES KO RASH-1C cells. (D) MSH2, MSH6, or EXO1 was knocked out in WT or HMCES KO RASH-1C cells, and cells induced with dox for 4 d were assayed for GFP loss. (E,F) Deletion frequencies in the HTS7 (E) and VDJ (F) regions from WT and HMCES KO RASH-1C cells with MSH2, MSH6, or EXO1 knocked out. (G,H) Distribution of deletion lengths in the HTS7 (G) and VDJ (H) regions from WT or HMCES KO RASH-1C cells with MSH2, MSH6, or EXO1 knocked out. 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.

Fig 5: Establishing a rapid assay for SHM. (A) Schematic of SHM reaction. During SHM, AID deaminates C to generate uracil, which can act as a template for replication, leading to a C:G-to-T:A transition mutation. Alternatively, noncanonical (error-prone) base excision repair (BER) initiated by UNG can create an abasic site that, if copied by a translesion DNA polymerase, yields mutations at G:C. Finally, U or U:G mismatches can trigger the generation of ssDNA gaps through the action of UNG and mismatch repair (MMR) factors MSH2, MSH6, and EXO1, which primarily yield mutations at A:T upon translesion polymerase synthesis. (B) Scheme for creating RASH-1 and RASH-2. Creation of RASH involved several steps of CRISPR/Cas9 gene targeting in Ramos. AICDA (encoding AID) was disrupted, and a construct was inserted into the AAVS1 safe harbor locus that expresses AID7.3 (AID hyperactive mutant with threefold increased catalytic activity) upon doxycycline (dox)-induced binding of Tet3G to a promoter containing seven copies of its binding site, TetO (7xTetO). Western blot showing AID protein levels in AID7.3in cells without or with 200 ng/mL dox for 24 h. The GFP7-E reporter with EF1α promoter driving transcription of HTS7 (enriched with AID hotspots)-T2A-GFP was integrated into IGH either 38,674 bp upstream of the V leader sequence (RASH-1) or by replacing IGH-VDJ coding sequences so as to retain the leader exon (which now lacks its ATG start codon; RASH-2). Kozak sequence was added to HTS7-T2A-GFP in RASH-2. (C,D) Representative flow cytometry plots of and bar graphs quantifying GFP loss (C) or IgM loss (D) in RASH-1 cells treated with dox for 6 d. (E) Representative flow cytometry plots of and bar graph quantifying GFP loss in RASH-2 treated with dox for 6 d. 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.