8536d_ch05_105-136 8/22/02 2: 47 PM Page 114 mac46 mac46: 1256_deh: 8536d: Goldsby et al./Immunology 5e 114 PART I Generation of B-Cell and T-Cell Response (a) Deletional joining (b) Inversional joining two signal sequences and the adjacent coding sequences a A reaction catalyzed by RAg-1 and RAG-2 in which the by RAG-1/2 and synapsis free 3-OH group on the cut DNA strand attacks the phosphodiester bond linking the opposite strand to the signal sequence, simultaneously producing a hairpin structure at the cut end of the coding sequence and a flush, 5-phosphorylated, double-strand break at the by RAG-1/2 Cutting of the hairpin to generate sites for the addition leonides, followed by the 3 few nucleotides from the coding sequence by a single- strand endonuclease ③ Hairpin formation a Addition of up to 15 nucleotides, called N-region and double- strand nucleotides, at the cut ends of the V, D, and J coding DNA break by sequences of the heavy chain by the enzyme terminal RAG- deoxynucleotidyl transferase Repair and ligation to join the coding sequences and to join the signal sequences, catalyzed by normal double- 和mmk strand break repair(DSBr)enzymes Recombination results in the formation of a coding joint, falling between the coding sequences, and a signal joint, be of p-nucleotides 非 tween the RSSs. The transcriptional orientation of the gene segments to be joined determines the fate of the signal joint and intervening DNA. When the two gene segments are in the same transcriptional orientation, joining results in dele tion of the signal joint and intervening DNA as a circular ex cision product(Figure 5-8). Less frequently, the two gene of N-nucleotides by tdT segments have opposite orientations. In this case joining oc- curs by inversion of the DNA, resulting in the retention of Coding joint to form joints by Gnetum rss Two-turn RSS FIGURES-7 Model depicting the general process of recombina tion of immunoglobulin gene segments is illustrated with Vx and Jk (a)Deletional joining occurs when the gene segments to be joined have the same transcriptional orientation(indicated by horizontal blue arrows). This process yields two products: a rearranged v) unit that includes the coding joint, and a circular excision product con- sisting of the recombination signal sequences(RSSs), signal joint, and intervening DNA. (b) Inversional joining occurs when the gene FIGURE 5-8 Circular DNA isolated from thymocytes in which the segments have opposite transcriptional orientations. In this case, the DNA encoding the chains of the T-cell receptor(TCR)undergoes re- RSSS, signal joint, and intervening DNA are retained, and the orien- arrangement in a process like that involving the immunoglobulin tation of one of the joined segments is inverted. In both types of re- genes. Isolation of this circular excision product is direct evidence for combination, a few nucleotides may be deleted from or added to the the mechanism of deletional joining shown in Figure 5-7.[From K. ends of the coding sequences before they are rejoin Okazaki et al., 1987, Cell 49: 477 Jtwo signal sequences and the adjacent coding sequences (gene segments) are brought into proximity ■ Cleavage of one strand of DNA by RAG-1 and RAG-2 at the junctures of the signal sequences and coding sequences ■ A reaction catalyzed by RAG-1 and RAG-2 in which the free 3-OH group on the cut DNA strand attacks the phosphodiester bond linking the opposite strand to the signal sequence, simultaneously producing a hairpin structure at the cut end of the coding sequence and a flush, 5-phosphorylated, double-strand break at the signal sequence ■ Cutting of the hairpin to generate sites for the addition of P-region nucleotides, followed by the trimming of a few nucleotides from the coding sequence by a single￾strand endonuclease ■ Addition of up to 15 nucleotides, called N-region nucleotides, at the cut ends of the V, D, and J coding sequences of the heavy chain by the enzyme terminal deoxynucleotidyl transferase ■ Repair and ligation to join the coding sequences and to join the signal sequences, catalyzed by normal double￾strand break repair (DSBR) enzymes Recombination results in the formation of a coding joint, falling between the coding sequences, and a signal joint, be￾tween the RSSs. The transcriptional orientation of the gene segments to be joined determines the fate of the signal joint and intervening DNA. When the two gene segments are in the same transcriptional orientation, joining results in dele￾tion of the signal joint and intervening DNA as a circular ex￾cision product (Figure 5-8). Less frequently, the two gene segments have opposite orientations. In this case joining oc￾curs by inversion of the DNA, resulting in the retention of 114 PART II Generation of B-Cell and T-Cell Responses (a) Deletional joining 5′ 3′ Vκ Jκ RSS 5′ 3′ Vκ Jκ (b) Inversional joining 3′ 5′ 3′ Recognition of RSSs by RAG-1/2 and synapsis L J Vκ κ Coding joint 5′ 3′ Signal joint Signal joint Coding joint Single-strand DNA cleavage by RAG-1/2 Hairpin formation and double-strand DNA break by RAG-1/2 Random cleavage of hairpin by endonuclease generates sites for the addition of P-nucleotides Optional addition to H-chain segments of N-nucleotides by TdT Repair and ligation of coding and signal sequences to form joints by DSBR enzymes 1 2 3 4 5 = Two-turn RSS = One-turn RSS L L + FIGURE 5-7 Model depicting the general process of recombina￾tion of immunoglobulin gene segments is illustrated with V and J. (a) Deletional joining occurs when the gene segments to be joined have the same transcriptional orientation (indicated by horizontal blue arrows). This process yields two products: a rearranged VJ unit that includes the coding joint, and a circular excision product con￾sisting of the recombination signal sequences (RSSs), signal joint, and intervening DNA. (b) Inversional joining occurs when the gene segments have opposite transcriptional orientations. In this case, the RSSs, signal joint, and intervening DNA are retained, and the orien￾tation of one of the joined segments is inverted. In both types of re￾combination, a few nucleotides may be deleted from or added to the cut ends of the coding sequences before they are rejoined. FIGURE 5-8 Circular DNA isolated from thymocytes in which the DNA encoding the chains of the T-cell receptor (TCR) undergoes re￾arrangement in a process like that involving the immunoglobulin genes. Isolation of this circular excision product is direct evidence for the mechanism of deletional joining shown in Figure 5-7. [From K. 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