P nucleotides . . ^ccoctAGAJaccc coding Joint or

N segment

D Jh

• • ssasaGGTCaleei: I . , ccoclCCAfilaccc coding join!

exonuclease deletion or D Jh

Figure 5 Formation of coding and signal joints. Heptamer fusion leads to an unmodified signal joint, and hairpin formation followed by exonuclease deletion and N and P nucleotide addition leads to a modified coding joint. (Reproduced with permission from Okada A and Alt FW (1995) The variable region gene assembly mechanism. In: Honjo T and Alt FW (eds) Immunoglobulin Genes. London: Academic Press.)

referred to as N-segment and palindromic trinucleotide additions. N-Segment diversity results from the addition of untemplated bases by terminal deoxynucleotidyl transferase (TdT). TdT activity is minimal in the fetus and neonate but is upregulated postnatally. As a result, the CDRH3 is shorter in the fetal compared with the adult repertoire. While TdT appears to act predominantly on H chains, N segments have been identified at L chain VJ junctions in about 20% of light chains. This indicates that TdT, or an analogous enzyme, must be active in a significant percentage of human B lymphocytes during L chain rearrangement. P Nucleotides are tem-plated bases from the noncoding strand in the coding joint as a result of the formation of hairpin structures generated during recombination. These usually consist of single bases or dinucleotides {Figure 5).

Not all of the proteins involved in this highly regulated DNA recombination event have been identified. Two proteins, RAG-1 and RAG-2, the products of the recombination activating genes RAG1 and RAG2, have signal-cutting activity. They are re quired for initiating V(D)J recombination. Studies indicate that there is synchronous cleavage at the two RSSs between the signal and the coding border. Cleavage is a two-step reaction: first, a single-strand nick is made at the 5' end of the heptamer, and secondly, a covalently closed hairpin coding end and a blunt 5' phosphorylated signal end are formed (Figure 5). Mutational analysis indicates that the recombination machinery must interact with both RSSs for cleavage to occur at either signal. Ku, a nuclear DNA repair protein, is also required for V(D)J recombination. Ku is a heterodimer composed of a 70 kDa and an 86 kDa subunit. Both subunits are required to form functional Ku. Ku binds DNA ends in a sequence-independent manner. The Kit heterodimer also associates with a DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to form a trimeric protein called DNA protein kinase (DNA-PK). DNA-PK has serine/threonine kinase activity and requires DNA for activation. One model suggests that following the cleavages by RAG-1 and RAG-2, Ku is then loaded on to the coding hairpin

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