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 required to mediate V-(D)-J joining. The RAG-1 and RAG-2 proteins 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

(a) Nucleotide sequence of RSSs CACAGTG-

GTGTCAC-

Heptamer

23 bp 23 bp

—ACAAAAACC

— TGTTTTTGG Nonamer

Two-turn RSS

C CAAAAACA— Nonamer

12 bp 12 bp

—CACTGTG

—GTGACAC Heptamer

One-turn RSS

(b) Location of RSSs in germ-line immunoglobulin DNA

L VK Jk

jh ch

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

RSS—designated one-turn RSS and two-turn RSS—have characteristic locations within X-chain, K-chain, and heavy-chain germline DNA. During DNA rearrangement, gene segments adjacent to 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.

(a) Deletional joining

L Vk

(b) Inversional joining

JK VK L JK

@ Single-strand DNA cleavage by RAG-1/2

@ Hairpin formation and double-strand DNA break by

RAG-1/2

(4 Random cleavage ' of hairpin by endonuclease generates sites for the addition of P-nucleotides ■'' ^

@ Hairpin formation and double-strand DNA break by

L VK JK Coding joint

® Optional addition to H-chain segments of N-nucleotides by TdT Repair and ligation of coding and 5 ^^^^— ~TI__LF3 signal sequences Signal —^

to form joints by joint Coding

DSBR enzymes joint

L VK JK Coding joint

Signal joint

FIGURE 5-7

Signal joint

FIGURE 5-7

Model depicting the general process of recombination of immunoglobulin gene segments is illustrated with VK 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 VJ unit that includes the coding joint, and a circular excision product consisting 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 orientation of one of the joined segments is inverted. In both types of recombination, a few nucleotides may be deleted from or added to the cut ends of the coding sequences before they are rejoined.

two 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 singlestrand 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 doublestrand break repair (DSBR) enzymes

Recombination results in the formation of a coding joint, falling between the coding sequences, and a signal joint, between 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 deletion of the signal joint and intervening DNA as a circular excision product (Figure 5-8). Less frequently, the two gene segments have opposite orientations. In this case joining occurs by inversion of the DNA, resulting in the retention of

Circular Dna Isolated From Thymocytes

FIGURE 5-8

Circular DNA isolated from thymocytes in which the DNA encoding the chains of the T-cell receptor (TCR) undergoes rearrangement 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. Okazaki et al., 1987, Cell 49:477.]

both the coding joint and the signal joint (and intervening DNA) on the chromosome. In the human k locus, about half of the Vk gene segments are inverted with respect to Jk and their joining is thus by inversion.

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