DNA Recombination:- Immunoglobulin Genes Assemble by Recombination
Some DNA rearrangements are a programmed part of development in eukaryotic organisms. An important ex ample is the generation of complete immunoglobulin genes from separate gene segments in vertebrate genomes. A human (like other mammals) is capable of producing millions of different immunoglobulins (anti bodies) with distinct binding specificities, even though the human genome contains only ~35,000 genes. Recom bination allows an organism to produce an extraordinary diversity of antibodies from a limited DNA-coding capacity. Studies of the recombination mechanism reveal a close relationship to DNA transposition and suggest that this system for generating antibody diversity may have evolved from an ancient cellular invasion of transposons. We can use the human genes that encode proteins of the immunoglobulin G (IgG) class to illustrate how antibody diversity is generated. Immunoglobulins con sist of two heavy and two light polypeptide chains . Each chain has two regions, a variable region, with a sequence that differs greatly from one immunoglobulin to another, and a region that is virtually constant within a class of immunoglobulins. There are also two distinct families of light chains, kappa and lambda, which differ somewhat in the sequences of their constant regions. For all three types of polypeptide chain (heavy chain, and kappa and lambda light chains), diversity in the variable regions is generated by a similar mechanism. The genes for these polypeptides are di vided into segments, and the genome contains clusters with multiple versions of each segment. The joining of one version of each of the segments creates a complete gene. Figure 25–44 depicts the organization of the DNA encoding the kappa light chains of human IgG and shows how a mature kappa light chain is generated. In undifferentiated cells, the coding information for this polypeptide chain is separated into three segments. The V (variable) segment encodes the first 95 amino acid residues of the variable region, the J (joining) segment encodes the remaining 12 residues of the variable region, and the C segment encodes the constant region. The genome contains ~300 different V segments, 4 different J segments, and 1 C segment. As a stem cell in the bone marrow differentiates to form a mature B lymphocyte, one V segment and one J segment are brought together by a specialized recombination system (Fig. 1). During this programmed DNA deletion, the intervening DNA is discarded. There are about 300 4 1,200 possible V–J combinations.
The recombination process is not as precise as the site-specific recombination described earlier, so additional variation occurs in the sequence at the V–J junction. This increases the overall variation by a factor of at least 2.5, thus the cells can generate about 2.5 1,200 3,000 different V–J combinations. The final joining of the V–J combination to the C region is accomplished by an RNA splicing reaction after transcription, a process described in Chapter 26. The recombination mechanism for joining the V and J segments is illustrated in Figure 2. Just beyond each V segment and just before each J segment lie re combination signal sequences (RSS). These are bound by proteins called RAG1 and RAG2 (recombination activating gene). The RAG proteins catalyze the formation of a double-strand break between the signal sequences and the V (or J) segments to be joined. The V and J segments are then joined with the aid of a second com plex of proteins.
FIGURE 1 Recombination of the V and J gene segments of the human IgG kappa light chain. This process is designed to generate antibody diversity. At the top is shown the arrangement of IgG-coding sequences in a bone marrow stem cell. Recombination deletes the DNA between a particular V segment and a J segment. After transcription, the transcript is processed by RNA splicing, as described in Chapter 26; translation produces the light-chain polypeptide. The light chain can combine with any of 5,000 possible heavy chains to produce an antibody molecule.
FIGURE 2 Mechanism of immunoglobulin gene rearrangement. The RAG1 and RAG2 proteins bind to the recombination signal sequences (RSS) and cleave one DNA strand between the RSS and the V (or J) segments to be joined. The liberated 3 hydroxyl then acts as a nucleophile, attacking a phosphodiester bond in the other strand to create a double-strand break. The resulting hairpin bends on the V and J segments are cleaved, and the ends are covalently linked by a com plex of proteins specialized for end-joining repair of double-strand breaks. The steps in the generation of the double-strand break cat alyzed by RAG1 and RAG2 are chemically related to steps in trans position reactions.
