The first reaction is an endonucleolytic cleavage that requires the coordinated activity of both RAG proteins. Initially, a complex of RAG-1 and RAG-2 proteins, together with high-mobility group chromatin protein HMGB1 or HMGB2, recognizes and aligns the two RSSs that are the target of the cleavage reaction. RAG-1 operates as a dimer, with RAG-2 acting as a cofactor (Fig. 5.9). RAG-1 specifically recognizes and binds the heptamer and the nonamer of the RSS and contains the Zn2+-dependent endonuclease activity of the RAG protein complex. As a dimer, RAG-1 seems to align the two RSSs that will undergo rearrangement. Recent models suggest that the 12/23 rule may be established because an essential asymmetric orientation of the RAG-1:RAG-2 complex induces a preference for binding to RSS elements of different types (Fig. 5.10). The bound RAG complex makes a single-strand DNA break at the nucleotide just 5ʹ of the heptamer of the RSS, thus creating a free 3ʹ-OH group at the end of the coding segment. This nucleophilic 3ʹ-OH group immediately attacks the phosphodiester bond on the opposite DNA strand, making a double-strand break and creating a DNA ‘hairpin’ at the coding region and a flush double-strand break at the end of the heptamer sequence. This cutting process occurs twice, once for the each gene segment being joined, producing four ends: two hairpin ends at the coding regions and two flush ends at both heptamer sequences (see Fig. 5.8). These DNA ends do not float apart, however, but are held tightly in the complex until a joining step has been completed. The blunt ends of the heptamer sequence are precisely joined by a complex of DNA ligase IV and XRCC4 to form the signal joint.
Formation of the coding joint is more complex. The two coding hairpin ends are each bound by Ku, which recruits the DNA-PKcs subunit. Artemis is recruited into this complex and is phosphorylated by DNA-PK. Artemis then opens the DNA hairpins by making a single-strand nick in the DNA. This nicking can happen at various points along the hairpin, which leads to sequence variability in the final joint. The DNA repair enzymes in the complex modify the opened
Shown as ribbon diagrams, the RAG-1:RAG-2 complex contains two RAG-1 (green and blue) and two RAG-2 proteins (purple). The first 383 amino acids of RAG-1 were truncated before crystallization. The N-terminal nonamer binding domain (NBD) of the two RAG-1 proteins undergoes domain swapping and mediates dimerization of the two proteins. The remainder of the RAG-1 protein contains the endonuclease activity that is dependent on the binding of a Zn2+ ion. Each RAG-1 protein binds a separate RAG-2 protein. Courtesy of Martin Gellert.
Zn2+ Zn2+ J region V region heptamer nonamer 23-bp RSS 12-bp RSS A 12-bp RSS bound to one RAG-1 favors binding of a 23-bp RSS to the other RAG-1
Fig. 5.10 The 12/23 base pair rule may result from asymmetric binding of RSSs to the RAG-1:RAG-2 dimer.