Recombination of gene segments containing recombination signal sequences (RSSs, triangles) begins with the binding of a complex of RAG-1 (purple), RAG-2 (blue), and high-mobility-group (HMG) proteins (not shown) to one of the RSSs flanking the coding sequences to be joined (second row). The RAG complex then recruits the other RSS. In the cleavage step, the endonuclease activity of RAG makes single-stranded cuts in the DNA backbone precisely between each coding segment and its RSS. At each cutting point this creates a 3ʹ-OH group, which then reacts with a phosphodiester bond on the opposite DNA strand to generate a hairpin, leaving a blunt double-stranded break at the end of the RSS. These two types of DNA ends are resolved in different ways. At the coding ends (left panels), essential repair proteins such as Ku70:Ku80 (green) bind to the hairpin. Ku70:80 forms a ringlike structure as a heterodimer, but the monomers do not encircle the DNA. The DNA-PK:Artemis complex (purple) then joins the complex, and its endonuclease activity opens the DNA hairpin at a random site, yielding either two flush-ended DNA strands or a single-strand extension. The cut end is then modified by terminal deoxynucleotidyl transferase (TdT, pink) and exonuclease, which randomly add and remove nucleotides, respectively (this step is shown in more detail in Fig. 5.11). The two coding ends are finally ligated by DNA ligase IV in association with XRCC4 (turquoise). At the signal ends (right panels), Ku70:Ku80 binds to the RSS but the ends are not further modified. Instead, a complex of DNA ligase IV:XRCC4 joins the two ends precisely to form the signal joint.
The other proteins in the recombinase complex are members of the ubiquitously expressed nonhomologous end joining (NHEJ) pathway of DNA repair known as double-strand break repair (DSBR). In all cells, this process is responsible for rejoining the two ends at the site of a double-strand break in DNA. The DSBR joining process is imprecise, meaning that nucleotides are frequently gained or lost at the site of joining. This has evolutionary relevance as in most cells it would not be advantageous to gain or lose nucleotides when repairing DSBs. However, in lymphocytes, the imprecise nature of DSBR is critical for junctional diversity and adaptive immunity. Thus, this may be the driving pressure for NHEJ to mediate imprecise joining. One ubiquitous protein contributing to DSBR is Ku, which is a heterodimer (Ku70:Ku80); this forms a ring around the DNA and associates tightly with a protein kinase catalytic subunit, DNA-PKcs, to form the DNA-dependent protein kinase (DNA-PK). Another protein that associates with DNA-PKcs is Artemis, which has nuclease activity. The DNA ends are finally joined together by the enzyme DNA ligase IV, which forms a complex with the DNA repair protein XRCC4. DNA polymerases μ and λ participate in DNA-end fill-in synthesis. In addition, polymerase μ can add nucleotides in a template-independent manner. In summary, lymphocytes have adapted several enzymes used in common DNA repair pathways to help complete the process of somatic V(D)J recombination that is initiated by the RAG-1 and RAG-2 V(D)J recombinases.