hairpins by removing nucleotides, while at the same time the lymphoidspecific enzyme terminal deoxynucleotidyl transferase (TdT), which is also part of the recombinase complex, adds nucleotides randomly to the single-strand ends. Addition and deletion of nucleotides can occur in any order, and one does not necessarily precede the other. Finally, DNA ligase IV joins the processed ends together, thus reconstituting a chromosome that includes the rearranged gene. This repair process creates diversity in the joint between gene segments while ensuring that the RSS ends are ligated without modification and that unintended genetic damage such as a chromosome break is avoided. Despite the use of some ubiquitous mechanisms of DNA repair, adaptive immunity based on the RAG-mediated generation of antigen receptors by somatic recombination seems to be unique to the jawed vertebrates, and its evolution is discussed in the last part of this chapter.
The in vivo roles of the enzymes involved in V(D)J recombination have been established through both natural and artificially induced mutations. Mice lacking TdT have about 10% of the normal level of non-templated nucleotides added to the joints between gene segments. This small remainder may result from the template-independent activity of DNA polymerase μ.
Mice in which either of the RAG genes has been inactivated, or which lack DNA-PKcs, Ku, or Artemis, suffer a complete block in lymphocyte development at the gene-rearrangement stage or make only trivial numbers of B and T cells. They are said to suffer from severe combined immune deficiency (SCID). The original scid mutation was discovered some time before the components of the recombination pathway were identified and was subsequently identified as a mutation in DNA-PKcs. In humans, mutations in RAG1 or RAG2 that result in partial V(D)J recombinase activity are responsible for an inherited disorder called Omenn syndrome, which is characterized by an absence of circulating B cells and an infiltration of skin by activated oligoclonal T lymphocytes. Mice deficient in components of ubiquitous DNA repair pathways, such as DNA-PKcs, Ku, or Artemis, are defective in double-strand break repair in general and are therefore also hypersensitive to ionizing radiation (which produces double-strand breaks). Defects in Artemis in humans produce a combined immunodeficiency of B and T cells that is associated with increased radiosensitivity. SCID caused by mutations in DNA repair pathways is called irradiation-sensitive SCID (IR-SCID) to distinguish it from SCID due to lymphocyte-specific defects.
Another genetic condition in which radiosensitivity is associated with some degree of immunodeficiency is ataxia telangiectasia, which is due to mutations in the protein kinase ATM (ataxia telangiectasia mutated), which are also associated with cerebellar degeneration and increased radiation sensitivity
Left panel: This cartoon of the structure shown in Fig 5.9 illustrates the flexibility of the hinge connecting the NBD to the catalytic domain of RAG-1. Right panel: the NBD domain of RAG-1 interacts with the RSS nonamer sequence (blue), while the RSS heptamer sequence (red) is bound to the portion of RAG-1 that contains the Zn2+ endonuclease activity. In this cartoon model, the interaction of a 12-bp RSS with one of the RAG-1 subunits induces the NBD domain to rotate toward the catalytic domain of RAG-1, to accommodate the length of the RSS. Since the two NBD domains are coupled by domain swaps, this induced conformation pulls the other NBD away from its RAG-1 subunit, which then prefers binding of the 23-bp RSS. The endonucleolytic cleavage (arrows) of the DNA by RAG-1 occurs precisely at the junction between the heptamer and the respective V, D, or J gene segment.