In heavy-chain gene rearrangements and in a proportion of human light-chain gene rearrangements, however, N-nucleotides are added by a quite different mechanism before the ends are rejoined. N-nucleotides are so called because they are non-template-encoded. They are added by the enzyme TdT to the single-stranded ends of the coding DNA after hairpin cleavage. After the addition of up to 20 nucleotides, single-stranded stretches may have some complementary base pairs. Repair enzymes then trim off nonmatching nucleotides, synthesize complementary DNA to fill in the remaining single-stranded gaps, and ligate the new DNA to the palindromic region (see Fig. 5.11). TdT is maximally expressed during the period in B-cell development when the heavy-chain gene is being assembled, and so N-nucleotides are common in heavy-chain V–D and D–J junctions. N-nucleotides are less common in light-chain genes, which undergo rearrangement after heavy-chain genes, when TdT expression has been shut off, as we will explain further in Chapter 8 when discussing the specific developmental stages of B and T cells.
Nucleotides can also be deleted at gene segment junctions. This is accomplished by exonucleases, and although these have not yet been identified, Artemis has dual endonuclease and exonuclease activity and so could well be involved in this step. Thus, a heavy-chain CDR3 can be shorter than even the smallest D segment. In some instances it is difficult, if not impossible, to recognize the D segment that contributed to CDR3 formation because of the excision of most of its nucleotides. Deletions may also erase the traces of P-nucleotide palindromes introduced at the time of hairpin opening. For this reason, many completed VDJ joins do not show obvious evidence of P-nucleotides. As the total number of nucleotides added by these processes is random, the added nucleotides often disrupt the reading frame of the coding sequence beyond the joint. Such frameshifts will lead to a nonfunctional protein, and DNA rearrangements leading to such disruptions are known as nonproductive rearrangements. As roughly two in every three rearrangements will be nonproductive, many B-cell progenitors never succeed in producing functional immunoglobulin and therefore never become mature B cells. Thus, junctional diversity is achieved only at the expense of considerable loss of cells during B-cell development. In Chapter 8, we return to this topic when we discuss the cellular stages of B-cell development and how they relate to the temporal sequence of rearrangement of the V, D, and J gene segments of the antigen receptor chains.
Summary.
The extraordinary diversity of the immunoglobulin repertoire is achieved in several ways. Perhaps the most important factor enabling this diversity is that V regions are encoded by separate gene segments (V, D, and J gene segments), which are brought together by a somatic recombination process— V(D)J recombination—to produce a complete V-region exon. Many different gene segments are present in the genome of an individual, thus providing a heritable source of diversity that this combinatorial mechanism can use. Unique lymphocyte-specific recombinases, the RAG proteins, are absolutely required to catalyze this rearrangement, and the evolution of RAG proteins coincided with the appearance of the modern vertebrate adaptive immune system. Another substantial fraction of the functional diversity of immunoglobulins comes from the imprecise nature of the joining process itself. Variability at the coding joints between gene segments is generated by the insertion of random numbers of Pand N-nucleotides and by the variable deletion of nucleotides at the ends of some segments. These are brought about by the random opening of the hairpin by Artemis and by the actions of TdT. The association of different lightand heavy-chain V regions to form the antigen-binding site of an immunoglobulin molecule contributes further diversity. The combination of all of these sources of diversity generates a vast primary repertoire of antibody specificities.
T-cell receptor gene rearrangement.