Mechanism and regulation of class switch recombination

Abstract

Antibody class switching occurs in mature B cells in response to antigen stimulation and costimulatory signals. It occurs by a unique type of intrachromosomal deletional recombination within special G-rich tandem repeated DNA sequences [called switch, or S, regions located upstream of each of the heavy chain constant (C(H)) region genes, except Cdelta]. The recombination is initiated by the B cell-specific activation-induced cytidine deaminase (AID), which deaminates cytosines in both the donor and acceptor S regions. AID activity converts several dC bases to dU bases in each S region, and the dU bases are then excised by the uracil DNA glycosylase UNG; the resulting abasic sites are nicked by apurinic/apyrimidinic endonuclease (APE). AID attacks both strands of transcriptionally active S regions, but how transcription promotes AID targeting is not entirely clear. Mismatch repair proteins are then involved in converting the resulting single-strand DNA breaks to double-strand breaks with DNA ends appropriate for end-joining recombination. Proteins required for the subsequent S-S recombination include DNA-PK, ATM, Mre11-Rad50-Nbs1, gammaH2AX, 53BP1, Mdc1, and XRCC4-ligase IV. These proteins are important for faithful joining of S regions, and in their absence aberrant recombination and chromosomal translocations involving S regions occur.

Figure 1

Diagram of Ig class switch recombination (CSR) to IgA. (a) The mouse IgH locus in B cells expressing IgM and IgD (by alternative RNA transcription/processing). During CSR, activation-induced cytidine deaminase (AID) deaminates dC residues in the top and bottom strands of transcriptionally active S regions (Sμ and Sα in the diagram shown), initiating a process (described in the text) that results in double-strand DNA breaks (DSBs) in both S regions and CSR by intrachromosomal deletion (b). (c) The IgH locus after CSR to IgA. Splicing diagrams of the μ, δ mRNAs and the germline α transcript are indicated below the diagram of the locus. Similar germline transcripts are induced from unrearranged Cγ,Cε, and Cα genes, depending on the cytokine stimulation received by the B cell.

Figure 2

Diagrams of (a) model for generation of DNA breaks, mutations, and translocations in IgS regions by AID-UNG-APE and (b) model for conversion of SSBs to DSBs by mismatch repair. (a) AID deaminates dC, resulting in dU bases, which are excised by one of the uracil DNA glycosylases, UNG. Abasic sites are cut by AP-endonuclease (APE1 and APE2) (74), creating SSBs that can spontaneously form DSBs if they are near each other on opposite DNA strands, or, if not, mismatch repair activity converts them to DSBs (see b). Alternatively, DNA Pol β can correctly repair the nick, preventing CSR, or error-prone translesion polymerases can repair the nick but introduce mutations. Finally, the DNA breaks can lead to aberrant recombinations/translocations. (b) AID is hypothesized to introduce several dU residues in S regions during one cell cycle. Some of the dU residues are excised by UNG, and some of the abasic sites are nicked by APE. The U:G mismatches that remain would be substrates for Msh2-–Msh6 (100). Msh2-–Msh6, along with Mlh1-Pms2, recruit Exo1 (and accessory proteins) to a nearby 5′ nick, from where Exo1 begins to excise toward the mismatch (90, 91), creating a DSB with a 5′ single-strand overhang, which can be filled in by DNA polymerase. Fill-in synthesis is probably performed by translesion polymerases owing to the presence of abasic sites. Alternatively, the overhang is removed by a 5′ flap endonuclease (Fen1) or by Exo1.

Figure 3

Proteins that bind to DSBs that are involved in CSR. The evidence supporting this diagram is discussed in the text. DNA-PK, which consists of Ku70–Ku80 and DNA-PKcs, most likely binds DSB ends first. Mre11-Rad50-Nbs1 (MRN) probably binds next, perhaps helping to hold the DNA ends together, and MRN recruits and activates ATM (ataxia telangiectasia mutated), which phosphorylates H2AX, 53BP1, and Mdc1, resulting in accumulation of large amounts of these proteins and MRN around the DSB sites. After the DNA ends are brought sufficiently near by activities of these other proteins, DNA-PK can hold the DNA ends in the correct position for recombination. These proteins are all important for correct S-S recombination, and they inhibit aberrant recombinations and translocations. ATM also phosphorylates proteins that regulate the cell cycle and induce apoptosis, but nothing is known about how important this is during CSR.