Lack Nonhomologous End Joining Recombination Events Occurring in B Cells and EXO 1 Are Important for Class - Switch

In the absence of core nonhomologous end-joining (NHEJ) factors, Ab gene class-switch recombination (CSR) uses an alternative end-joining (A-EJ) pathway to recombine switch (S) region DNA breaks. Previous reports showing decreased S-junction micro-homologies in MSH2-deficient mice and an exonuclease 1 (EXO1) role in yeast microhomology-mediated end joining suggest that mismatch repair (MMR) proteins might influence A-EJ–mediated CSR. We have directly investigated whether MMR proteins collectively or differentially influence the A-EJ mechanism of CSR by analyzing CSR in mice deficient in both XRCC4 and individual MMR proteins. We find CSR is reduced and that Igh locus chromosome breaks are reduced in the MMR/XRCC4 double-deficient B cells compared with B cells deficient in XRCC4 alone, suggesting MMR proteins function upstream of double-strand break formation to influence CSR efficiency in these cells. Our results show that MLH1, EXO1, and MSH2 are all important for efficient A-EJ–mediated CSR, and we propose that MMR proteins convert DNA nicks and point mutations into dsDNA breaks for both C-NHEJ and A-EJ pathways of CSR. We also find Mlh1-XRCC4 2 B cells have an increased frequency of direct S junctions, suggesting that MLH1 proteins may have additional functions that influence A-EJ–mediated CSR. B cells switch from the production of IgM Abs to other classes of Abs (IgG, IgE, or IgA) by the process of class-switch recombination (CSR); CSR alters the Ab effector function without changing the Ag specificity. Ab effector functions are encoded in the constant gene segments located within the IgH (Igh) locus. DNA breaks are generated in switch (S) regions located upstream of constant gene segments and then joined through long-range recombinational events, bringing a downstream constant gene segment into close proximity with the Ag-specific V region gene segments, to generate different Ab isotypes (1). One factor required for the generation of DNA breaks in S regions is activation-induced cytidine deaminase (AID) (2). AID deaminates cytidine residues to uracil residues generating U-G nucleotide mispairings in S-region sequences (2–7). Current models suggest that AID U-G mispairings are recognized by DNA repair factors such as mismatch repair (MMR) and base excision repair (BER) proteins, which, in turn, are important for generating dsDNA breaks in S regions (8–14). In the absence of each of the individual MMR proteins, MSH2, MSH6, MLH1, PMS2, or exonuclease 1 (EXO1), CSR is reduced ∼2-to 5-fold compared with wild-type (15–19). Previous studies have shown that U-G mispairings are targets of the MMR pathway (20–22), …