To elucidate which function of APE1 is important for CSR, we expressed APE1 WT and its loss-of-function mutant proteins in and and Table S1), and the mutation base profile remained unchanged (Table S2), indicating that APE1 is not required for 5 S mutation

To elucidate which function of APE1 is important for CSR, we expressed APE1 WT and its loss-of-function mutant proteins in and and Table S1), and the mutation base profile remained unchanged (Table S2), indicating that APE1 is not required for 5 S mutation. Open in a separate window Fig. recombination 11 homolog (MRE11) and carboxy-terminal Acta2 binding protein (CtBP)-interacting protein (CtIP), are responsible for the remaining CSR activity in the absence of APE1. reduces the CSR efficiency in CH12F3-2A cells to 20% of the wild-type (WT) cells, whereas a deletion of APE2 has no effect on the CSR of CH12F3-2A cells (35). The results clearly demonstrated the involvement of APE1 in CSR, but at the same time raised several critical questions as to the role of APE1 in CSR. First, it is of particular importance to determine with which enzymatic activity and by what mechanism APE1 is involved in CSR. It is also important to assess whether APE1 is also required for AID-induced SHM. Furthermore, it is interesting to know which enzymes could account for the remaining CSR activity in APE1-deficient CH12F3-2A cells, although Masani et al. proposed that a latent endonuclease activity of the MMR factor MLH1/PMS2 complex may be responsible (35). In the present study, we examined APE1s role in CSR and SHM using APE1-deficient CH12F3-2A cells (35) and found that, although APE1s endonuclease activity is required for CSR, it is dispensable for SHM and IgH/c-myc translocation. Surprisingly, the endonuclease activity of APE1 is dispensable for AID-induced S-region cleavage, but necessary for Ku80 recruitment and synapse formation of the broken ends. Our results suggest that APE1 functions as a DNA end resection enzyme and plays a critical role in processing AID-induced SSBs for efficient joining and recombination during CSR. Results The Endonuclease Activity of APE1 Is Required for CSR. To elucidate which function of APE1 is important for CSR, we expressed APE1 WT and its loss-of-function mutant proteins in and and Table S1), and the mutation base profile remained unchanged (Table S2), indicating that APE1 is not required for 5 S mutation. Open in a separate window Fig. 2. APE1 is SK1-IN-1 dispensable for AID-induced 5 S mutation. values (Fishers exact test) for significant difference are shown in the graph. The detailed results are shown in Tables S1 and S2. (and and and and values (Fishers exact test) for significant difference are shown in the graph. (and em B /em ). Furthermore, the accumulation of Ku80, a protein critical for NHEJ, was SK1-IN-1 very much reduced at S regions of vector- SK1-IN-1 and Y170F-transfectant cells compared with WT transfectant (Fig. 5 em C /em ), indicating that the reduced CSR in vector and Y170F transfectants might be due to the less efficient generation of DSBs with blunt ends. Open in a separate window Fig. 5. APE1 is required for efficient SCS synapse formation during CSR. ( em A /em ) Scheme of long-range interactions between SCS elements in the IgH locus before and after AID activation. ( em B /em ) Representative gel picture of the 3C assay detecting SCS interaction in the three cell lines stimulated (or not) with CIT for 24 h. GAPDH was amplified as loading control. ( em C /em ) ChIP and quantitative PCR analysis for Ku80 in cells stimulated (or not) with CIT for 24 h. Data are represented as mean SD. APE1 May Function as SK1-IN-1 Cleaved-End Processing Enzyme for Ig Diversification. Although AID-induced S-region cleavage takes place normally in APE1 deficiency, both Ku80 accumulation and the synapse formation of the broken ends are severely affected by the absence of the endonuclease activity of APE1. We speculated that APE1 is involved in 3 end processing of SSBs during CSR, because it is well established that the APE1 is involved in the 3 end processing of SSBs (30, 40, 41). To test this possibility, we investigated whether other broken end-processing enzymes are responsible for the residual CSR activity in APE1-deficient CH12F3-2A cells. siRNA knockdown or drug inhibition of meiotic recombination 11 homolog (MRE11) could significantly reduce the remaining CSR activity in the absence of APE1 (Fig. 6 em A /em ). Such reduction was more robust in case of CtIP knockdown (Fig. 6 em B /em ). Open in a separate window Fig. 6. The involvement of end-processing enzymes in the residual switching in em Ape1 /em -null CH12F3-2A cells. ( em A /em , em B /em , and em D /em ) Protein expression ( em Upper /em ) and IgA switching efficiency ( em Lower /em ) of em Ape1 /em -null CH12F3-2A cells transfected with the indicated siRNA oligos and stimulated with CIT for 24 or 48 h. ( em C /em ) Relative IgA switching efficiency (bar graph) and cell viability (dot plot) of CH12F3-2ACBcl2 cells treated with various concentrations of the proteasome inhibitor Bortezomib. In all datasets,.