We have demonstrated that AID binds cooperatively with both UNG and Msh2-Msh6 to the Ig Sμ region in B cells induced to switch to IgG3, and that the cooperative binding requires the C terminal 10 amino acids of AID. Full-length AID was not detected at Sμ by ChIP in cells lacking UNG, Msh2, or Msh6 and thus all three components are required for complex formation. The interactions might be direct or indirect. Consistent with the ChIP results, we found that the MMR proteins Msh2 and Msh6 do not contribute to CSR in cells expressing ΔAID, indicating that the ability of AID to recruit MMR proteins to Sμ is important for the role of MMR during CSR. The associations between AID and UNG and between AID and Msh2-Msh6 are detected when AID is localized on S regions; we have not been able to detect binding in the absence of DNA or at the Cμ region. The binding to both Sμ and Sγ3 are only detected when AID is competent for deaminase activity, suggesting that the cooperative binding between AID and UNG and Msh2-Msh6 occurs after AID has deaminated dC's. However, as not all dU's are likely to be excised by UNG and converted to SSBs, it is likely that at any one moment in cells undergoing CSR, S regions contain a mixture of dU's, abasic sites, and SSBs (1
). Thus, S regions could have substrates for UNG and MMR binding, despite also having SSBs and DSBs.
It is puzzling that there are about as many Sμ DSBs in cells expressing ΔAID as in cells expressing full-length AID, despite lower recruitment of AID, UNG and MMR. Considerable evidence indicates that UNG is essential for S region DSBs (16
), and that MMR is important for conversion of SSBs to DSBs in S regions during CSR. In the absence of MMR, DSBs appear to only form from SSBs that are near on opposite strands (19
). Sμ region DSBs have also been observed using LM-PCR in HIGM2 patients with C terminal deletions of AID (48
). Also, CH12F3-2 lymphoma B cells expressing C terminal deleted AID have been shown to have as many Sμ region DSBs as cells expressing full length AID (25
). Therefore, it seems clear that in the absence of the AID C terminus, both UNG and MMR can still access dU and U:G mismatches resulting from AID activity. One possibility to explain the numerous DSBs detected in cells expressing ΔAID is that ΔAID might have higher catalytic activity than full-length AID (27
), and the increased dU's might require fewer UNG and MMR molecules to induce DSBs. However, we favor the alternative possibility that DSBs are induced with lower efficiency in cells expressing ΔAID, but are also repaired or recombined inefficiently and thus accumulate. This appears consistent with the fact that GFP-low cells expressing ΔAID have fewer DSBs than GFP-low cells expressing full-length AID. We hypothesize that in GFP-high cells this repair problem would be exacerbated due to overwhelming the repair/recombination mechanisms because of the high AID levels, thus explaining the similar numbers of DSBs observed in ΔAID- and AID-expressing GFP-high and in total GFP+ cells. Inefficient repair/recombination is also consistent with the increased translocations found in B cells expressing RV-ΔAID (25
), as unresolved DSBs increase the risk of DNA translocations, carcinogenesis, and cell death.
Our results suggest that the C terminus of AID promotes the formation of a protein complex that not only generates S region DNA breaks, but also promotes their repair in a manner that leads to productive CSR. UNG and MMR proteins might help recruit other proteins involved in subsequent repair steps during CSR. For example, Msh2 and Msh6 have been found in a large complex of DNA repair proteins in HeLa cells, termed BASC, which also includes Mre11-Rad50-Nbs1 (MRN). MRN binds DSBs induced by AID and is known to be important for repair of DSBs and for CSR (1
). Another possibility is suggested by findings in over-expression experiments that AID binds DNAPKcs, dependent upon the AID C terminus (53
), and that Msh2-Msh6 binds to Ku70-Ku80 (54
). As Ku and DNA-PKcs are important for directing DNA breaks towards NHEJ, it is possible that in addition to its role in converting SSBs to DSBs, MMR associated with the AID C terminus is involved in recruiting DNA-PK-Ku proteins to sites of AID-induced DSBs. Chromosomal translocations have been shown to be mediated by microhomology-mediated end joining (55
), an alternative form of end-joining in which recombination is mediated by short microhomologies between the two recombining sequences. In addition, B cells with mutations in DNA damage response or repair genes that are involved in CSR, including mutations in MMR or UNG genes, often show increased lengths of junctional microhomology (46
). Most importantly, Sμ-Sα junctions from two human patients expressing AID with C terminal deletions show highly significantly increased lengths of junctional microhomology (59
). These results suggest that the AID C terminus, MMR and UNG are involved in the recombination step during CSR and perhaps direct S region DSBs towards NHEJ.
CSR and V-D-J recombination both involve NHEJ and occur during G1 phase of the cell cycle. Recent studies have shown that a mutant of RAG-2 (T490A), which prevents degradation of RAG-2 during S phase, results in aberrant V-D-J recombination and increased chromosomal translocations, indicating the importance of cell cycle control for accurate recombination (60
). AID-dependent Sμ DSBs are only observed during G1 phase in normal splenic B cells undergoing CSR, indicating that most DBS are introduced, recombined and repaired during G1 phase (21
). It is possible that the interaction of AID with UNG and MMR might be important to recruit these proteins to S regions during G1 phase, since UNG and MMR proteins are primarily associated with S phase activity. MMR is known to associate with the replication machinery and to correct nucleotides mis-incorporated during S phase due to DNA polymerase errors (23
). UNG is important for removal of dU incorporated instead of dT during replication, and in both primary fibroblasts and HeLa cells UNG is expressed at much higher levels during S phase than during G1 or G2 phases (63
). However, in splenic B cells induced to undergo CSR, UNG expression is slightly higher in G1 phase than in S phase. UNG is active in G1 phase B cell extracts, and is clearly important for excision of dUs due to deamination by AID (14
). It is possible that if UNG and MMR are not specifically recruited to Sμ during G1 phase by full-length AID, these proteins might only be available at S regions during S phase, thus creating DSBs in S phase. During DNA replication, DSBs cause stalling of DNA polymerase and collapse of replication forks, which are then sometimes repaired by break-induced replication, which is highly inaccurate (65
). This could lead to translocations and greatly reduced CSR. Alternatively, inefficient repair of DSBs in G1 phase cells expressing ΔAID could cause DSBs to be retained during S and G2 phases, and thus lead to chromosomal rearrangements.
The binding of UNG and MMR to AID might be indirect and occur via other proteins. AID has been shown to interact with several proteins (66
). Protein kinase A (PKA), which phosphorylates AID at S38, is specifically localized at S regions during CSR, and might participate in a PKA-RPA-AID complex (67
). Phosphorylation of AID at S38 is required for interaction with RPA and for CSR (69
), but it is possible that the AID C terminus is also involved. Interestingly, RPA32 has been shown to interact with UNG by a yeast two-hybrid assay (71
). Also, a recent publication reported that the C-terminus of AID interacts with the 14-3-3 adaptor protein complex and that this interaction is important for recruitment of AID to S regions, as 14-3-3 binds specifically to AGCT motifs (72
). Deficiency in 14-3-3 proteins results in a 50% decrease in CSR. However, since the ΔAID-ER mutant induces as many Sμ mutations as AID-ER, the recruitment of AID by 14-3-3 is not necessary for the ability of AID to deaminate Sμ. Also, we find that the preferential occurrence of Sμ DSBs at AGCT hotspots is identical in cells expressing both AID and ΔAID. It is possible, however, that 14-3-3, when bound to AID, is important for recruiting UNG and MMR and other repair proteins to Sμ.