8536d_ch05_105-136 8/22/02 2: 47 PM Page 113 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immunology 5e Organization and Expression of Immunoglobulin Genes CHAPTER 5 113 spacer In heavy-chain DNA, the signal sequences of the VH Mechanism of Variable-Region and JH gene segments have two-turn spacers, the signals on DNA Rearrangements either side of the DH gene segment have one-turn spacers (Figure 5-6b) Signal sequences having a one-turn spacer can Now that we've seen the results of variable-region gene re- join only with sequences having a two-turn spacer(the so- arrangements, let's examine in detail how this process occurs called one-turn/two-turn joining rule). This joining rule en- during maturation of B cells. sures,for example, that a Vi segment joins only to a JL segment and not to another Vi segment; the rule likewise en Recombination Signal Sequences sures that VH, DH, and JH segments join in proper order and Direct recombination that segments of the same type do not join each other. The discovery of two closely related conserved sequences in Gene Segments Are Joined by Recombinases derstanding of the mechanism of gene rearrangements. DNA -(D)-)recombination, which takes place at the junctions sequencing studies revealed the presence of unique recombi- between RSSs and coding sequences, is catalyzed by enzymes nation signal sequences(RSSs)flanking each germ-line V, collectively called v(D) recombinase D, and J gene segment. One RSS is located 3 to each V gene Identification of the enzymes that catalyze recombination segment, 5,to each gene segment, and on both sides of each of V, D, and gene segments began in the late 1980s and is still D gene segment. These sequences function as signals for the ongoing. In 1990 David Schatz, Marjorie Oettinger, and recombination process that rearranges the genes. Each RSS David Baltimore first reported the identification of two contains a conserved palindromic heptamer and a conserved recombination-activating genes, designated RAG-Iand AT-rich nonamer sequence separated by an intervening se- RAG-2, whose encoded proteins act synergistically and are re- quence of 12 or 23 base pairs (Figure 5-6a). The intervening quired to mediate V-(D)-1joining. The RAG-1 and RAG-2 pro- 12-and 23-bp sequences correspond, respectively, to one and teins and the enzyme terminal deoxynucleotidyl transferase are the only lymphoid-specific gene products that called one-turn recombination signal sequences and two. have been shown to be involved in V-(D)-)rearrangement. The recombination of variable-region gene segments The Vk signal sequence has a one-turn spacer, and the k consists of the following steps, catalyzed by a system of re- gnal sequence has a two-turn spacer In A light-chain DNA, mbinase enzymes(Figure 5-7) this order is reversed; that is, the va signal sequence has a Recognition of recombination signal sequences(RSSs) wo-turn spacer, and the Jx signal sequence has a one-turn by recombinase enzymes, followed by synapsis in which (a) Nucleotide sequence of RSSs GTGTCAC-23 bpFTGTTTTTGG CCAAAAACA-12bpFGTGACAC Heptamer Nonamer Nonamer Heptamer Two-turn RSS One-turn rss b) Location of RSSs in germ-line immunoglobulin DNA K-chain DNA … h Heavv- chain DNA5′ …◆…“}3 FICURE5-6 Two conserved sequences in light-chain and heavy. RSS-designated one-turn RSS and two-turn RSS-have charac chain DNA function as recombination signal sequences(RSSs). teristic locations within A-chain, K-chain, and heavy-chain germ- (a)Both signal sequences consist of a conserved palindromic hep- line DNA During DNA rearrangement, gene segments adjacent to tamer and conserved AT-rich nonamer; these are separated by the one-turn RSS can join only with segments adjacent to the two- nonconserved spacers of 12 or 23 base pairs. (b) The two types of turn RSS.Mechanism of Variable-Region DNA Rearrangements Now that we’ve seen the results of variable-region gene re￾arrangements, let’s examine in detail how this process occurs during maturation of B cells. Recombination Signal Sequences Direct Recombination The discovery of two closely related conserved sequences in variable-region germ-line DNA paved the way to fuller un￾derstanding of the mechanism of gene rearrangements. DNA sequencing studies revealed the presence of unique recombi￾nation signal sequences (RSSs) flanking each germ-line V, D, and J gene segment. One RSS is located 3 to each V gene segment, 5 to each J gene segment, and on both sides of each D gene segment. These sequences function as signals for the recombination process that rearranges the genes. Each RSS contains a conserved palindromic heptamer and a conserved AT-rich nonamer sequence separated by an intervening se￾quence of 12 or 23 base pairs (Figure 5-6a). The intervening 12- and 23-bp sequences correspond, respectively, to one and two turns of the DNA helix; for this reason the sequences are called one-turn recombination signal sequences and two￾turn signal sequences. The V signal sequence has a one-turn spacer, and the J signal sequence has a two-turn spacer. In  light-chain DNA, this order is reversed; that is, the V signal sequence has a two-turn spacer, and the J signal sequence has a one-turn spacer. In heavy-chain DNA, the signal sequences of the VH and JH gene segments have two-turn spacers, the signals on either side of the DH gene segment have one-turn spacers (Figure 5-6b). Signal sequences having a one-turn spacer can join only with sequences having a two-turn spacer (the so￾called one-turn/two-turn joining rule). This joining rule en￾sures, for example, that a VL segment joins only to a JL segment and not to another VL segment; the rule likewise en￾sures that VH, DH, and JH segments join in proper order and that segments of the same type do not join each other. Gene Segments Are Joined by Recombinases V-(D)-J recombination, which takes place at the junctions between RSSs and coding sequences, is catalyzed by enzymes collectively called V(D)J recombinase. Identification of the enzymes that catalyze recombination of V, D, and J gene segments began in the late 1980s and is still ongoing. In 1990 David Schatz, Marjorie Oettinger, and David Baltimore first reported the identification of two recombination-activating genes, designated RAG-1 and RAG-2, whose encoded proteins act synergistically and are re￾quired to mediate V-(D)-J joining. The RAG-1 and RAG-2 pro￾teins and the enzyme terminal deoxynucleotidyl transferase (TdT) are the only lymphoid-specific gene products that have been shown to be involved in V-(D)-J rearrangement. The recombination of variable-region gene segments consists of the following steps, catalyzed by a system of re￾combinase enzymes (Figure 5-7): ■ Recognition of recombination signal sequences (RSSs) by recombinase enzymes, followed by synapsis in which Organization and Expression of Immunoglobulin Genes CHAPTER 5 113 FIGURE 5-6 Two conserved sequences in light-chain and heavy￾chain DNA function as recombination signal sequences (RSSs). (a) Both signal sequences consist of a conserved palindromic hep￾tamer and conserved AT-rich nonamer; these are separated by nonconserved spacers of 12 or 23 base pairs. (b) The two types of RSS—designated one-turn RSS and two-turn RSS—have charac￾teristic locations within -chain, -chain, and heavy-chain germ￾line DNA. During DNA rearrangement, gene segments adjacent to the one-turn RSS can join only with segments adjacent to the two￾turn RSS. (a) Nucleotide sequence of RSSs CACAGTG GTGTCAC 23 bp 23 bp ACAAAAACC TGTTTTTGG Heptamer Nonamer Two-turn RSS 12 bp 12 bp Nonamer One-turn RSS Heptamer CACTGTG GTGACAC GGTTTTTGT CCAAAAACA (b) Location of RSSs in germ-line immunoglobulin DNA 5′ 3′ VH Heavy-chain DNA DH JH CH 5′ 3′ Vκ κ-chain DNA Jκ Cκ 5′ 3′ Vλ λ-chain DNA J L λ Cλ L L 8536d_ch05_105-136 8/22/02 2:47 PM Page 113 mac46 mac46:1256_des:8536d:Goldsby et al. / Immunology 5e: