SUMMARY Activation Induced cytidine Deaminase (AID) initiates Immunoglobulin (Ig) heavy chain (IgH) class switch recombination (CSR) and Ig variable region somatic hypermutation (SHM) in B lymphocytes by deaminating cytidines on template and non-template strands of transcribed DNA substrates. However, the mechanism of AID access to the template DNA strand, particularly when hybridized to a nascent RNA transcript, has been an enigma. We now implicate the RNA exosome, a cellular RNA processing/degradation complex, in targeting AID to both DNA strands. In B-lineage cells activated for CSR, the RNA exosome associates with AID, accumulates on IgH switch regions in an AID-dependent fashion, and is required for optimal CSR. Moreover, both the cellular RNA exosome complex and a recombinant RNA exosome core complex impart robust AID- and transcription-dependent DNA deamination of both strands of transcribed SHM substrates in vitro. Our findings reveal a role for non-coding RNA surveillance machinery in generating antibody diversity.

In the absence of core nonhomologous end-joining (NHEJ) factors, antibody 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 microhomologies in MSH2-deficient mice, and an exonuclease1 (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 to 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 double strand DNA breaks for both C-NHEJ and A-EJ pathways of CSR. We also find Mlh1-XRCC4- B cells have an increased frequency of direct S-junctions, suggesting that MLH1 proteins may have additional functions that influence A-EJ mediated CSR.

The Msh2 mismatch repair (MMR) protein is critical for class switch recombination (CSR) events that occur in mice that lack the Sμ tandem repeat (SμTR) region (SμTR−/− mice). The pattern of microhomology among switch junction sites in Msh2-deficient mice is also dependent on the presence or absence of SμTR sequences. It is not known whether these CSR effects reflect an individual function of Msh2 or the function of Msh2 within the MMR machinery. In the absence of the SμTR sequences, Msh2 deficiency nearly ablates CSR. We now show that Mlh1 or Exo1 deficiencies also eliminate CSR in the absence of the SμTR. Furthermore, in SμTR−/− mice, deficiencies of Mlh1 or Exo1 result in increased switch junction microhomology as has also been seen with Msh2 deficiency. These results are consistent with a CSR model in which the MMR machinery is important in processing DNA nicks to produce double-stranded breaks, particularly in sequences where nicks are infrequent. We propose that double-stranded break paucity in MMR-deficient mice leads to increased use of an alternative joining pathway where microhomologies are important for CSR break ligation. Interestingly, when the SμTR region is present, deficiency of Msh2 does not lead to the increased microhomology seen with Mlh1 or Exo1 deficiencies, suggesting that Msh2 might have an additional function in CSR. It is also possible that the inability to initiate MMR in the absence of Msh2 results in CSR junctions with less microhomology than joinings that occur when MMR is initiated but then proceeds abnormally due to Mlh1 or Exo1 deficiencies.

Summary Immunoglobulin (Ig) class switch recombination (CSR) occurs most often by intrachromosomal recombinations between switch (S) regions located on a single chromosome, but can also occur by interchomosomal recombinations between Ig heavy chain (Igh) S regions located on chomosomal homologs. Interchromosomal recombinations have also been found between chromosomes that are not homologs; examples are Igh/c-myc and Igh/transgene translocations. Most, but not all, studies have indicated that activation-induced cytidine deaminase (AID) is important in Igh/c-myc translocations. The role of AID has not been determined for Igh/transgene translocations. We now show that the majority of Igh/transgene translocations between non-homologs from an Ig transgenic mouse are dependent on AID, but we also find a small number of these translocations that can occur in the absence of AID. Surprisingly, our results also indicate that, even though Sγ switch sequences in the endogenous Igh locus participate in chromosomal translocations with the non-homolog transgene-bearing chromosome, Sμ switch sequences do not. This contrasts with the fact that both endogenous Sμ and Sγ sequences participate in intrachromosomal CSR. Our findings suggest the operation of a regulatory mechanism that can differentially control the accessibility of Sμ and Sγ regions for non-homolog translocations even when both are accessible for intrachromosomal recombination.