Fig. 5.7 V-region gene segments are joined by recombination. Top panel: in every V-region recombination event, the recombination signal sequences (RSSs) flanking the gene segments are brought together to allow recombination to take place. The 12-bp-spaced RSSs are shown in orange, the 23-bp-spaced RSSs in purple. For simplicity, the recombination of a light-chain gene is illustrated; for a heavy-chain gene, two separate recombination events are required to generate a functional V region. Left panels: in most cases, the two segments undergoing rearrangement (the V and J gene segments in this example) are arranged in the same transcriptional orientation in the chromosome, and juxtaposition of the RSSs results in the looping out of the intervening DNA. Recombination occurs at the ends of the heptamer sequences in the RSSs, creating the so-called signal joint and releasing the intervening DNA in the form of a closed circle. Subsequently, the joining of the V and J gene segments creates the coding joint in the chromosomal DNA. Right panels: in other cases, the V and J gene segments are initially oriented in opposite transcriptional directions. In this case, alignment of the RSSs requires the coiled configuration shown, rather than a simple loop, so that joining the ends of the two heptamer sequences now results in the inversion and integration of the intervening DNA into a new position on the chromosome. Again, the joining of the V and J segments creates a functional V-region exon.
direction of transcription L2 V2 Vn Ln L2 V2 L1 V1 JL1 V1 direction of transcription JLn Vn deleted inverted signal joint coding joint After recombination this loop is excised from the chromosome, taking the two RSS regions with it After recombination the coiled region is retained in the chromosome in an inverted orientation L1 V1 L2 L2 V2 V2 23 23 23 12 L2 V2 Vn Vn Ln Ln J J L1 V1 J Ln Vn L1 V1 V gene segments may be in either forward or reverse transcriptional orientation relative to downstream gene segments When a forward-oriented V gene segment recombines with a downstream gene segment, alignment of the two RSS regions loops out the intervening DNA When a reverse-oriented V gene segment recombines with a downstream gene segment, alignment of the RSS regions forms the intervening DNA into a coiled conÿguration
The complex of enzymes that act in concert to carry out somatic V(D)J recombination is termed the V(D)J recombinase. The lymphoid-specific components of the recombinase are called RAG-1 and RAG-2, and they are encoded by two recombination-activating genes, RAG1 and RAG2. This pair of genes is essential for V(D)J recombination, and they are expressed in developing lymphocytes only while the lymphocytes are engaged in assembling their antigen receptors, as described in more detail in Chapter 8. Indeed, the RAG genes expressed together can confer on nonlymphoid cells such as fibroblasts the capacity to rearrange exogenous segments of DNA containing the appropriate RSSs; this is how RAG-1 and RAG-2 were initially discovered.
DNA ligase IV:XRCC4 ligates DNA ends Ku70:Ku80 binds DNA ends Precise signal joint DNA ligase:XRCC4 Signal joints Ku80 Ku70 5´-phosphorylated blunt ends TdT processes DNA ends Ku70:Ku80 binds DNA ends DNA ligase IV:XRCC4 ligates DNA ends terminal deoxynucleotidyl transferase (TdT) DNA ligase:XRCC4 1223 JV RAG-1:2 binds RSS Synapsis of two RSSs Germline confguration Cleavage of RSSs Coding joints DNA-PK:Artemis opens hairpin RAG-1/2 Ku70 Ku80 Artemis DNA-PK covalently closed DNA hairpin ends Imprecise coding joint
Fig. 5.8 Enzymatic steps in RAG-dependent V(D)J rearrangement.