the new constant-region gene. In such a recombination event, the Cμ coding regions and the entire intervening DNA between Cμ and the S region undergoing rearrangement are deleted. Figure 10.21 illustrates switching from Cμ to Cε in the mouse. All switch recombination events produce genes that can encode a functional protein, because the switch sequences lie in introns and therefore cannot cause frameshift mutations.
The enzyme AID initiates class switch recombination, and acts only on regions of DNA being transcribed. Certain properties of the switch region sequences
Fig. 10.22 RNA processed from switch region introns interacts with AID and guides its activity. Top panel: promoters upstream of each switch region initiate transcription by RNA polymerase upstream of a rearranged VH gene, as in the case of Cμ, shown here, or a noncoding exon for all other constant regions. In all cases, the switch region itself lies within an intron upstream of the exons encoding the constant regions. This intronic switch region RNA is removed from the primary RNA transcript by splicing at specific splice acceptor and donor sites. Middle panel: after splicing, the switch region RNA is further processed and its repetitive elements allow the formation of putative G-quadruplex structures. Evidence indicates that these RNAs are able to bind AID, as implied in the cartoon. Bottom panel: the RNA acts as a guide to bring AID to the switch region by the ability of the G-quadruplex to hybridize with the original DNA template strand from which it was transcribed.
promote the accessibility to AID when they are being transcribed. Each switch region consists of many repeats of a G-rich sequence element on the non-template strand. For example, Sμ consists of about 150 repeats of the sequence (GAGCT)n(GGGGGT), where n is usually 3 but can be as many as 7. The sequences of the other switch regions (Sγ, Sα, and Sε) differ in exact sequence but all contain repeats of the GAGCT and GGGGGT sequences. It appears that movement of RNA polymerase through this highly repetitive region is occasionally halted—called polymerase stalling. This may be caused by bubble-like structures, called R-loops, that form when the transcribed RNA displaces the non-template strand of the DNA double helix (see Fig. 10.21, third panel) due to having many G residues in tandem on one strand.
Polymerase stalling seems closely connected with the recruitment of AID to specific switch regions being transcribed. A multisubunit RNA processing/ degradation complex, the RNA exosome, associates with AID and accumulates on transcribed switch regions, and the protein Spt5 associates with the stalled polymerase; both are necessary for AID to generate double-stranded breaks. Recent evidence indicates that AID is selectively guided to the transcribed switch by an additional mechanism. After an RNA polymerase has completed transcription of one RNA template, the intron RNA harboring the switch region is spliced out. This RNA is processed to generate an RNA structure, called a G-quadruplex, that is based on the G-rich repetitive element of the switch region (Fig. 10.22). This G-quadruplex serves a dual purpose, both binding to AID and also associating with the switch region from which it was transcribed, based on its sequence complementarity. Thus the G-quadruplex guides AID to the appropriate switch region, where particular palindromic sequences, such as AGCT, act as good substrates to allow its cytidine deaminase activity to act on both strands concurrently. In this way, the G-quadruplex functions in a manner similar to the synthetic guide RNAs that deliver the Cas9 endonuclease to specific genomic regions, as described in Appendix I, Section A-35).