It is important to recognize that the pattern of 12and 23-bp spacers used by the various gene segments is different between the λ, κ, and heavy-chain loci (see Fig. 5.6). Thus, for the heavy chain, a DH gene segment can be joined to a JH gene segment and a VH gene segment to a DH gene segment, but VH gene segments cannot be joined to JH gene segments directly, as both VH and JH gene segments are flanked by 23-bp spacers. However, they can be joined with a DH gene segment between them, as DH segments have 12-bp spacers on both sides (see Fig. 5.6).
In the antigen-binding region of an immunoglobulin, CDR1 and CDR2 are encoded directly in the V gene segment (see Fig. 5.2). CDR3 is encoded by the additional DNA sequence that is created by the joining of the V and J gene segments for the light chain, and the V, D, and J gene segments for the heavy chain. Further diversity in the antibody repertoire can be supplied by CDR3 regions that result from the joining of one D gene segment to another D gene segment, before being joined by a J gene segment. Such D–D joining is infrequent and seems to violate the 12/23 rule, suggesting that such violations of the 12/23 rule can occur at low frequency. In humans, D–D joining is found in approximately 5% of antibodies and is the major mechanism accounting for the unusually long CDR3 loops found in some heavy chains.
The mechanism of DNA rearrangement is similar for the heavyand light-chain loci, although only one joining event is needed to generate a light-chain gene but two are required for a heavy-chain gene. When two gene segments are in the same transcriptional orientation in the germline DNA, their rearrangement involves the looping out and deletion of the DNA between them (Fig. 5.7, left panels). By contrast, when the gene segments have opposite transcriptional orientations, the rearrangement retains the intervening DNA in the chromosome but with an inverted orientation (see Fig. 5.7, right panels). This mode of recombination is less common, but it accounts for about half of all Vκ to Jκ joins in humans because the orientation of half the Vκ gene segments is opposite to that of the Jκ gene segments.
5-5 The reaction that recombines V, D, and J gene segments modifying enzymes.
The overall enzymatic mechanisms involved in V-region rearrangement, or V(D)J recombination, are illustrated in Fig. 5.8. Two RSSs are brought together by interactions between proteins that specifically recognize the length of the spacers and thus enforce the 12/23 rule for recombination. The DNA molecule is then precisely cleaved by endonuclease activity at two locations and is then rejoined in a different configuration. The ends of the heptamer sequences are joined in a head-to-head fashion to form a signal joint. In the majority of cases, no nucleotides are lost or added between the two heptamer sequences, creating a double-heptamer sequence 5ʹCACAGTGCACAGTG3ʹ within the DNA molecule. When the joining segments are in the same orientation, the signal joint is contained in a circular piece of extrachromosomal DNA (see Fig. 5.7, left panels), which is lost from the genome when the cell divides. The V and J gene segments, which remain on the chromosome, join to form what is called the coding joint. When the joining segments are in the opposite relative orientation to each other within the chromosome (see Fig. 5.7, right panels), the signal joint is also retained within the chromosome, and the region of DNA between the V gene segment and the RSS of the J gene segment is inverted to form the coding joint. This situation leads to rearrangement by inversion. As we shall see later, the coding joint junction is imprecise, meaning that nucleotides can be added or lost between joined segments during the rearrangement process. This imprecise nature of coding joint formation adds to the variability in the V-region sequence, called junctional diversity.
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