V(D)J recombination of immunoglobulin genes

Adv. Biophys., Vol. 31, pp. 163-180 (1995)

V(D)J RECOMBINATION OF IMMUNOGLOBULIN GENES

CHIO OKA AND MASASHI KAWAICHI

Division of Gene Function in Animals, Nara Institute of Sci- ence and Technology, Ikoma, Nara 630-01, Japan

During lymphocyte differentiation, complete immunoglobulin (Ig)

and T cell receptor (TCR) genes are assembled from V (variable), J (joining), and, in some cases, D (diversity) gene segments by a site- specific somatic DNA rearrangement (1-3). This process, known as V(D)J recombination, is one of the strategies by which the immune system acquires diversity in recognition .of a tremendous number of antigens. The V, D, and J segments are flanked by recombination signal sequences each of which consists of a highly conserved palindromic heptamer and an AT rich nonamer separated by a non- conserved spacer sequence of either 12 or 23 base pairs. V(D)J recombination occurs significantly only between two signals with different spacer lengths (the 12/23 joining rule) (4). The initial events of the V(D) J recombination may be recognition of the signals and cleavage of DNA strands just outside the heptamers (Fig. 1). The heptamers are joined tail-to-tail to form a signal joint. The other two ends are joined to make a coding joint in a manner different from the signal joint formation, i.e., the coding joint formation usually accompanies loss of several nucleotides at both ends and addition of a new nucleotide sequence (N sequence (I, 5) and P nucleotides (6)) between them. This modification of the coding joints contributes further to the generation of diversity in antigenic recognition. Although the changes of DNA structure during V(D)J recombination have been fully described, the

163

164 C. OKA AND M. KAWAICHI

Singnal joint

Site-specific double-strand break

Coding joint

Fig. 1. Schema of V(D)J recombination. The first step of V(D)J recombination is

the formation of a double strand break at the end of heptamers in recombination

signals. This step seems to be catalyzed by lymphoid-specific mechanisms. The

next step generates two distinctive joints, a coding joint and a signal joint. The

formation of the joints may be catalyzed by a general DNA repair system involved

in double-strand DNA repair.

biochemical mechanism in V(D) J recombination has not been analyzed in detail.

V(D)J recombination is the only site-specific somatic recombination known to occur in cells of vertebrates. This recombination is a highly regulated process. The time of onset and shut-off of the recombination are strictly controlled during lymphocyte maturation. V(D) J recombination proceeds in a fairly strict order: first D to J and V to DJ of the Ig heavy chain genes, then V to J of the Ig K chain genes and, finally, V to J of the Ig 3L chain genes. Successful rearrangement of one allele of the Ig heavy chain genes seems to inhibit rearrangement of the other allele so that a single B-cell can produce only one type of antibody (allelic exclusion) (7-9). In contrast, the successful rearrangement of the heavy chain gene induces rearrangement of the Ig light chain genes (10). When a B-cell succeeds in rearrangement of a K chain gene, the recombination machinery will be shutoff to avoid further rearrangement of A chain genes, so that a single B-cell produces a single type of light chain (isotypic K-A exclusion).

The paucity of knowledge on reaction mechanisms, the complex but strict regulation, and its importance in medical and biological fields make V(D)J recombination a very intriguing subject.

I. ANALYSIS OF TRANSGENIC MICE CARRYING ARTIFICIAL SUBSTRATE

FOR V(D)J RECOMBINATION

To make an animal model for analysis of V(D)J recombination in oiz~o,

V(D)J RECOMBINATION 16.5

we constructed transgenic mice bearing an artificial substrate for this recombination (I 2). The artificial substrate plasmid, pAccat, contains human interleukin 2 receptor light (IL-2R L) chain cDNA in the inverted orientation relative to the upstream chicken p-actin promoter (Fig. 2). The cDNA segment is flanked by Ig recombination signals so that the segment can be inverted upon V(D)J-type recombination. When the cDNA is inverted, the human IL-2R L chain is expressed on the cell surface. The p-actin promoter is highly active in a wide range of cell types (ZZ), so that the V(D)J-type recombinase activity

4 4.0 Kb m

(4 p -actin promoter human IL-2R SV40 splice and

and intron JK L chain cDNA VK ~01~ (A)

p -actin promoter

(b) p-actin promoter Ex 1 V I( lad Ex2 J K icE1 CK KE2

Ex 1

Fig. 2. Structures of artificial recombination substrates. (a) Structure of pAccat

(II). (b) Structure of the substrate used by Matsuoka et al. (19). This DNA con-

tains whole VK and JK segments, instead of synthetic signal oligomers used in

pAccat, as recombination signals. It also contains splicing donor and acceptor

sequences (Ex 1 and Ex 2) of the /3-actin gene. The presence of these splicing

signals allows imprecise recombination with deletion up to 2 kb of DNA.


can be easily detected in cells or tissues by staining with anti-human IL-2R antibody (13).

The pAccat transgenic mice grew normally. pAccat turned out to be an excellent substrate for V(D)J recombination; the human IL-2R L chain was expressed in approximately 50% of cells in the spleen and 70% of cells in the thymus of an 8-week old transgenic mouse. South- ern blot hybridization of DNA obtained from the spleen and thymus of

the transgenic mice clearly demonstrated the expected DNA rearrangement of pAccat DNA. Neither surface staining with anti-human IL-2R antibody nor rearranged DNA bands on Southern blot analysis could be detected in the liver or other tissues such as the brain, kidney, lung, skeletal, muscle, or testis. To confirm that the inversion actually occurred through V(D) J recombination, DNA fragments containing the recombination junctions were amplified by polymerase chain reaction (PCR), cloned and sequenced. The sequencing of the PCR products containing the signal joints showed that the two heptamers were joined precisely tail to tail, and no loss or addition of nucleotides was detected in any case. On the other hand, the coding joints of the transgenic spleen and thymus cells lost several nucleotides from either

or both of the two ends. The N sequence insertion was demonstrated at the coding joints. These data showed that pAccat functioned as an authentic V(D) J recombination substrate in viva

The most intriguing argument in V(D) J recombination has been whether non-lymphoid tissues contain a recombination activity similar to that in lymphocytes (14). We analyzed rearrangement of the transgene in non-lymphoid tissues of transgenic mice by immunohis- tochemical staining with antibodies against the human IL-2R L chain and by in situ hybridization. The expression of the human IL-2R L chain was not detected in non-lymphoid tissue by either method. To further examine recombination of the artificial substrate in non-lymphoid tissues, we carried out highly sensitive “double PCR” amplification. In this PCR, one rearranged molecule among a hundred thousand unrearranged molecules was detected. Rearrangement was detected in all tissues examined. The amplified rearranged bands were most prominent in the lung and least in the brain or testis among non- lymphoid tissues. This pattern seemed to correlate with the amount of lymphocyte distribution in organs. By transplanting bone marrow cells of the transgenic mice into normal irradiated mice, it was demonstrated that the rearranged bands detected in non-lymphoid tissues of the transgenic mice were due to contamination of migrating lymphocytes. We concluded that non-lymphoid tissues including the central nervous

V(D)J RECOMBINATION 167

system did not contain the recombination activity with the same speci-

ficity as the immune system. It had long been speculated that the site-specific somatic recom-

bination similar to V(D)J recombination could occur in the central

nervous system to promote the diversification in functions of numerous neurons. This speculation was based on some phenomenological similarities between immune system and the central nervous system, such as extreme complexity and capacity for memory (14). In addition, a number of transcripts of Ig superfamily genes have been found in the central nervous system (15). Some of them, for instance, Thy-l and CD-4, are co-expressed in both the brain and T lymphocytes in mouse (16,17). RAG-l, which plays an essential role in V(D)J recombination, is also expressed at a low level in the central nervous system (18). Matsuoka et al. constructed transgenic mice carrying a V(D)J recombination substrate quite similar to ours (Fig. 2) (19). Their substrate contained the bacterial P-galactosidase gene (lad) as a reporter gene. The lad gene was similarly placed in an inverted orientation relative to the transcriptional direction of the chicken /!l-actin enhancer-promoter complex. Rearrangement can be detected by sensitive histo- chemical staining with X-gal (producing a blue color). In the adult spleens, the frequencies of blue-colored cells were lc)-4 to lo-* of unstimulated splenocytes, frequencies much lower than those found in the pAccat transgenic mice. By PCR amplification of the junctions followed by sequencing of the products, they confirmed that their substrate was rearranged in lymphocytes with the same specificity as that of V(D)J recombination. Surprisingly, they demonstrated that a wide variety of anatomical loci in the central nervous system of the

transgenic mouse were stained blue. By PCR amplification of the brain DNA, they detected multiple DNA fragments containing recombination break points, although the sizes of the fragments were different from the size anticipated from V(D)J recombination of the transgene. Sequencing of the PCR products showed that the recombination break points were not adjacent to the recombination signals but located various distances away from the signals. They concluded that the brain had an imprecise but V(D)J-1 k i e recombination activity. There are, however, at least two other possible interpretations of their data (Z&22). One is that the PCR products were derived from artificial recombination during PCR amplification in vitro. They showed that the recombination break points in the brain contained homologies of 2 to 9 nucleotides at the junctions. Such homologies seem to be sufficient for artificial “PCR mediated recombination”. Another possibility is that the


blue cells observed in the brain result from backward transcripts from a cryptic promoter within the transgene or near the transgene integra- tion site. The frequency of blue cells in the brain was apparently much higher than that expected from the amounts of the PCR products. There was discordance between the frequency of blue cells in the brain and that in the lymphocytes.

The adult mice deficient in the RAG-l gene showed normal development of the brains and normal behavior (23). The RAG-l de- pendent V(D) J recombination activity, therefore, is not essential for the function of the central nervous system.

It is quite difficult to exclude the presence of a somatic recombination activity in the central nervous system. But it may be clear that this system does not contain the recombination activity with the same specificity as that in the immune system. To clarify the issue of somatic recombination in the central nervous system (or in other tissues), some

different approaches may be necessary, such as cloning and analysis of circular DNA in the brain (24-26) or identification of genes inverted or deleted during the development of the brain, using a sophisticated method like the one utilizing in-gel competitive reassociation (27).

II. RBP-JIG AS A RECOMBINATION SIGNAL BINDING PROTEIN

The results with various transgenic mice and plasmid substrate for V(D)J recombination indicated that the signal sequences are sufficient to allow the DNA to be rearranged by V(D)J recombinase (II, 19, 28- 31). V(D)J recombinase should recognize the signal DNA sequences and probably forms a stable complex with the signals.

Hamaguchi et al. first purified a DNA binding protein specific to a V(D)J recombination signal sequence from 38B9, which is a mouse pre-B cell line active in V(D)J recombination (32). In their paper, they examined the DNA binding specificity of this protein using several DNA probes derived from the V(D)J recombination signals. The result suggested that this protein specifically bound to the recombination signal with a 23-base pair spacer (JK type signal) but did not to the recombination signal with a 12-base pair spacer (VK type signal), hence it was called RBP-JK ( recombination signal binding protein, JK type

specific). This protein did not bind to a mutated J~type signal probe with a point mutation in the heptamer at the third nucleotide from the end distal to the nonamer. The region between the first and the third nucleotides of the heptamer in the V(D) J recombination signals is most highly conserved. They also examined the presence of JK type signal


w

Fig. 3. Amino acid sequences of Drosophila and mouse RBP-JK proteins and

comparison of integrase motifs. (a) Upper lines marked d show the sequence of the

Drosophila protein. Lower lines show the sequence of the mouse protein. Only

amino acid residues different from the Drosophila protein are shown for the mouse

protein. Integrase motifs are indicated by a box. (b) Integrase motifs of the human

(h), mouse (m), and Drosophila (d) RBP-JK proteins.

sequence binding activity in nuclear extracts from various mouse tissues and culture cells. The activity similar to the RBP-JKprotein was detected only in the nuclear extract prepared from lymphoid cells.

Based on the partial amino acid sequences of the purified RBP- JK protein, Matsunami et al. cloned the full length cDNA of mouse RBP- J K (33). The amino acid sequence predicted from the cDNA showed that this protein consisted of 524 amino acids (Fig. 3). It did not show any homology to various domains of known DNA binding proteins, but extensive homology search in databases revealed the presence of a 40-amino acid motif in the middle of the protein. The motif is common to a group of site specific recombinases known as the integrase family (34, 35). The integrase family includes il phage integrase, fimbriae switch recombinase of Escherichia co& yeast Flp recombinase, and other recombinases found in plasmid, phage, and bacteria. Among the 40-amino acids of the integrase motif, four are known to be highly conserved in all members of the integrase family. Those residues are


the first histidine, the fourth arginine, the 38th tyrosine, and the 40th histidine. The mouse RBP-J~protein retained the first, the 3&h, and the 40th amino acid residues, but the fourth arginine was replaced by glycine. Studies on h integrase and yeast Flp indicated that the 38th tyrosine reacted with a phosphate group at the end of the cleaved DNA to form a protein-DNA conjugate as an intermediate of the recombination reaction (36, 37). On the basis of the DNA binding specificity and the presence of the integrase motif, the RBP-JKprotein was considered to be a candidate as a component of V(D)J recombinase.

The purified mouse RBP- JKprotein did not show activity related to recombination such as DNA cleavage activity or topoisomerase activity. But during the purification procedures, a DNA ligase activity was reported to be copurified with the RBP-Jrcprotein (38). It might be possible that the RBP- JK protein formed a complex with the ligase activity in viva.

III. UBIQUITOUS DISTRIBUTION OF RBP-JK

Subsequently, Hamaguchi et al. showed the ubiquitous presence of both RBP-JKmRNA and its protein in mouse tissues and various cell lines (38). The level of expression was almost constant in most cells. When genomic DNA from various species were analyzed by Southern blotting, Furukawa et al. found that the RBP-JK gene was present in various animals from Drosophila to human (39). The Drosophila and human RBP-JK genes were cloned by Furukawa et al. (39) and Amakawa and his colleagues, respectively (40). The mouse and human RBP- JKproteins were 98% identical at the amino acid level. The amino acid sequences of the Drosophila and mouse proteins diverged consid- erably in the N-terminal and C-terminal regions with 73% identity along the entire region of the proteins (Fig. 3). When the 250 amino acid in the middle of the RBP- Jlcprotein was compared, the mouse and Drosophila RBP- JK proteins were 93% identical and could be aligned without any gaps. The presence of the RBP-JKgene in the invertebrate and high conservation of this gene between Drosophila and mouse, along with the ubiquitous expression of the gene in adult mouse tissues, suggested a function of the RBP-JK gene other than V(D)J recombination.

The human RBP-JKgene (IGKJRB) was located at chromosome 3q25. Human has two pseudogenes located at chromosomes 9~13 and 9q13 (40); these pseudogenes were of the processed type lacking in- trons. The human RBP-JK gene was separated into at least 11 exons


which spanned 67 kb or more. Similarly, the mouse RBP-JK gene spanned at least 50 kb with 11 exons (41). Three or two transcripts with different 5’ structures were produced from the human or mouse RBP-Jicgene, respectively, by alternative splicing in viva (40, 41). The significance of the alternative splicing is not clear.

IV. DROSOPHILA HOMOLOGUE OF RBP-JK

The presence of the RBP-Jlcgene in Drosophila gave us a good oppor- tunity to analyze the function of RBP- JK by virtue of the well devel- oped genetics in Drosophila. The Drosophila RBP- Jrcgene was found to locate in the region of 35BC on chromosome 2 by in situ hybridization (39). This region was characterized in detail by Ashburner and his co-

workers (42, 43). Numerous deletion mutants and almost saturating numbers of recessive lethal mutants were available in this region. Quantitative Southern blot analysis of genomic DNAs from a number of mutant flies with deficiency in this region narrowed down the loca- tion of the RBP-J~gene to an approx. 50 kb region where only two recessive lethal genes had been located. Because of the high conservation of the RBP-JK genes among animals, recessive lethality was expected. The sequencing of the RBP-JK gene of several alleles (point mutations and PM mutagenized) of the two recessive lethal mutations proved that actually one mutation, known as the Suppressor of Hairless (L%(H)), was caused by mutations of the RBP-JKgene (44, 45).

1’. FUNCTION OF RBP-JK IN DROSOPHILA

Hairless (H) and Su(Erl) antagonistically regulate the development of the peripheral nervous system in Drosophila (42). In an early stage of this development, a cluster of neuroectodermal cells gain the potential to become nerve ceils by the action of proneural genes (Fig. 4) (46). Then, a single cell in the cluster is somehow selected to become a sensory mother cell. The differentiation into sensory organ cells of other cells surrounding the sensory mother cell is inhibited by the mechanism known as lateral inhibition. A number of neurogenic genes, such as Delta, Notch, Enhancer of Split (E(spl)), and so on, function in this singling-out stage. In a later stage of the development of sensory bristles, a sensory mother cell divides once to make two precursor cells. One precursor cell divides again and makes hair (trichogen) and socket (tormogen) cells. The other precursor cell also divides and makes a neuron and a glia (thecogen) cell. Heterozygous loss-of-function muta-


External sensoryprgan

cuticle-

dendrit

Prepaltern genes

a-p axis d-v axis

PPXleUral genes

daughtedess achaete-scute

complex

Neurogenic genes

Notch Delta Enhancer of

split neuralized mastermind bigbrain

Neuronal precursor type Selector genes

cut rhomboid

Cell division and lineage genes

cyclin numb oversensitive

Fig. 4. A model of development of the Drosophila peripheral nervous system.

Upper figure shows structure of the external sensory organ (46). A sensory bristle

is composed of four cells: two supporting cells (hair and socket cells), a neuron, and a glia. These four cells are derived from a single sensory mother cell as shown in the lower figure. Lower figure shows a model of development of peripheral

nervous system in Drosophila according to ref. 46. After determination of the body segments, proneural genes confer a cluster of cells in a segment with potential to

become nerve cells. From the cluster, a single sensory mother cell is selected by the

function of neurogenic genes. The sensory mother cell divides twice to differenti- ate into the four cells of the bristle.

tions of H show two distinct phenotypes on the sensory bristles of the adult fly; bristle loss phenotype and double socket phenotype (47). Severe loss of H function affects the early stage and causes the failure of specification of sensory mother cells, so that mechanosensory bristles fail to appear. Partial loss of H function results in a later defect, a nearly complete transformation of the trichogen cell into a second tormogen cell, yielding double socket phenotype. Heterozygous loss- of-function mutations of RBP- JK (Su(I-2)) suppress and gain-of-function mutations enhance the H phenotypes (42,45). Thus, Su(rr) and H function at two distinct developmental stages: at the stage of specification of sensory mother cells and later at the stage of cell fate determination of a precursor cell into trichogen and tormogen cells. One should


note that homozygous loss-of-function mutations of Su(H) and H are both lethal in late larval to early pupal stages. Su(H) must, therefore, have essential functions in a wide range of tissues including the peripheral nervous system.

The H gene has been cloned (48, 49); it encodes a novel basic protein with a molecular mass of 109 kD. The amino acid composition of the H protein is strongly skewed with alanine, serine, and proline comprising 40% of the H sequence. The predicted structure of the H protein did not show any motif homologous to known proteins except for a short segment which is similar to the PRD repeat motif (48). The PRD repeat is present in a number of homeodomain proteins and other transcription factors in Drosophila as well as in higher vertebrates. The presence of the PRD motif may suggest that the H protein functions as a transcription factor, although experimental evidence suggesting the nuclear localization, DNA binding activity, or transcriptional activity of the H protein has been lacking.

Genetical observation that H mutation suppresses the neural hyperplasia resulting from mutations of various neurogenic genes such as Delta, Notch, neuralixed, mastermind but not E(spl) has led to the suggestion that E(spl) may be the major target of the H function and hence of the SU(~ function (50, 51). The E(spl) gene cluster encodes several nuclear proteins containing basic-Helix-Loop-Helix motifs (52,

53). WPO, H, and S’u(H) apparently function downstream of Notch which encodes a transmembrane protein working as a signal-transduc- ing receptor for lateral inhibition (46, 54). Mutation of Notch has a pleiotropic effect, suggesting that the Notch signal transduction system functions in various Drosophila tissues. It is probable that the signal from Notch receptor is transferred to Hand/or Su(H) and they in turn regulate the expression of E(spZ). N 0 somatic rearrangement of E(spl) or other neurogenic genes is known to date (52, 53).

VI. DNA BINDING SPECIFICITY OF RBP-Jr PROTEIN

The DNA binding characteristics of the mouse RBP-JK protein was analyzed in detail by Tun et al. (55). By enriching RBP-JK-bound oligonucleotides from a pool of random oligonucleotides, they found that the RBP-JK protein recognizes a consensus sequence of CGTGGGAA, a sequence partly overlapping with the heptamer of V(D) J recombination signals (CACTGTG) but not exactly the same (Fig. 5). This consensus motif is similar to the binding sequence of NF-KB (56). A similar sequence was also found in the promoter region


Consensus sequence of RBP-JK binding

a g/cCGTGGGAA a/c

. . :

,*.

*I .I

*.

INTEGRASE MOTIF

I ‘I

*. *;’

I , **. I I

*se’ 526

I I _I I I I I I t

Spl I Dra I Pvu II Kpn I Hind Ill Rsa I Apa I Rsa I Pst I

Fig. 5. Consensus binding sequence and DNA binding domain of the mouse

RBP-JK protein. The mouse RBP-JK protein recognizes a consensus sequence of CGTGGGAA along with a few nucleotides on both sides of this core sequence.

The DNA binding domain was determined by assaying DNA binding activity of various mutant RBP-J~proteins. Open circles show position of mutations without

significant changes in DNA binding activities. Closed circles show mutations with markedly decreased DNA binding activities. Shaded circles indicate mutations

with marginal effects.

of Drosophila m8 gene in the E(spl) gene cluster (52). The upstream region (up to -900 bp) of the WZ~ gene contains at least 6 N-box sequences to which a group of basic-Helix-Loop-Helix proteins can bind. The RBP-JKbinding motif is approx. 600 bases upstream from the transcription start site of the m8 gene. The oligonucleotide probe derived from the m8 RBP- JK binding motif bound to the mouse RBP- JK protein at least 5 fold better than the V(D) J recombination signal sequence.

VII. DNA BINDING DOMAIN OF RBP-JK PROTEIN

Chung et al. introduced many mutations into the mouse RBP- JK protein, expressed the mutant proteins in Cos cells, and measured DNA binding activity of the mutant proteins to locate the DNA binding domain in the RBP-Jrcprotein (57). The RBP-JKprotein requires strict three dimensional structure for its stability. Slight deletions in either the N-terminal, the C-terminal, or the middle of the protein dimin-


ished the expression of the protein to undetectable levels. This instabil-

ity prevented us from positively locating the DNA binding domain. Results with point mutations indicated that the region between 212K and 230H was important for DNA binding (Fig. 5). This region is just in front of the integrase motif (230H-269H) and within the most highly conserved region of the RBP-JK protein; only one amino acid among 47 residues in this region is different between Drosophila and mouse RBP-Jrcproteins (33, 39). The region between 212K and 230H of the mouse RBP- Jlcprotein probably constitutes the DNA binding domain.

Drosophila RBP-Jrcmutants induced by ethyl methanesulfonate (EMS) treatment gave rise to a gain-of-function mutant (Enhancer of Hairless) as well as loss-of-function Su(H) mutants (42). The gain-of-function mutant, S5, had a point mutation in the putative DNA binding domain and, as expected, the S5 protein did not bind the DNA probes (44). One weak loss-of-function mutant, HG36, had a point mutation in the integrase motif and retained the DNA binding activity, indicating that the integrase motif of the RBP-J~protein has some physiologi- cal function (44). Although the data on these mutant proteins cannot be fit into a comprehensible scheme at present, they may suggest that the RBP-Jrcprotein interacts with other proteins in a complex manner.

RBP-JK was identified as a protein binding specifically to the V(D)J recombination signals and originally thought to be a candidate for a component of V(D)J recombinase (32, 33). But our studies revealed that this protein regulates the development of various tissues, especially the peripheral nervous system, in Drosophila (39, 44, 45). Recent data on target mice of the RBP-Jrcgene revealed that this gene is essential in early stages of mouse embryonal development. The mouse RBP- Jrcprotein like Drosophila homologue most probably functions as a transcriptional regulator of genes involved in the differentiation of tissues. On the analogy of the Drosophila system, it is probable that mouse RBP- JK functions in the pathway of signal transduction through Notch. Mouse has at least three genes homologous to the Dros- ophila Notch gene (58-61). Recent gene-targeting of mouse Notch 1 showed that the Notch 1 gene is also essential in postimplantation development of mouse (62).

VIII. GENES AND PROTEINS INVOLVED IN V(D)J RECOMBINATION

Several proteins and genes have been reported to be involved in V(D)J recombination (Table I). The recombination proceeds in two steps (Fig. 1); first, lymphoid specific machinery recognizes a V(D)J recom-


bination signal, cleaves DNA in the vicinity of the signal, and holds the cleaved DNA ends until they are properly processed by an exonuclease and terminal deoxynucleotidyl transferase; second, general DNA repair machinery joins the ends in a new combination so that the new configuration of the DNA is established.

RAG-l and RAG-2 together can confer V(D)J recombination activity on non-lymphoid cells and are components of the lymphoid specific machinery (63, 64). Mutant mouse in which either RAG-l or RAG-2 is target-disrupted lacks mature lymphocytes due to the failure of V(D) J recombination (23, 65). They are co-expressed at a significant level essentially only in lymphoid tissues active in V(D)J recombination (63, 64). Discordant expression of RAG-l and RAG-2 was reported in some tissues (18, 66). Low levels of RAG-l are expressed in the mouse central nervous system (18). RAG-2 is expressed in the absence of RAG-l expression in the chicken bursa of Fabricius which is active in gene conversion of the immunoglobulin genes (66). The function of RAG-2 in gene conversion was once suggested, but a later experiment showed that targeted disruption of RAG-2 in the bursal B cells did not affect the rate of gene conversion (67). The significance of the discordant expression is not clear.

Both RAG-l and RAG-2 are highly conserved in vertebrates. The C-terminal half of the RAG-l protein shows a homology to the yeast HPRl gene product, which is homologous to yeast DNA topoisomerase I (68, 69). Mutation in HPRl results in an increase in the rate of homologous, intrachromosomal deletional recombination

TABLE I

V(D)J recombination-related factors and genes Activity Reference

RAG-1 and RAG-2

scid gene

xrs-1 gene

XR-1 gene

Endonucleolytic activity

Nonamer binding protein

Heptamer binding protein

Recombination sequence-

binding protein

Induction of recombination in

non-lymphoid cells

Recombination and general DNA repair



Double strand break near

recombination signals

Specific recognition of

nonamer sequence

Specific recognition of heptamer in

VFi signals

Binding to both 12- and 23-spacer signals

63, 64

72

76

76

77, 78, 79

80, 81

82, 83

84


(69). The N-terminal half of the RAG-l protein contains a cysteine- histidine motif which is similar to the zinc-finger motif of a number of DNA binding proteins. When deletions were introduced in the RAG- 1 polypeptide, the C-terminal half was shown to be essential for recombination activating activity but the N-terminal half ‘containing the zinc-finger motif was dispensable (70). These data suggest that RAG- 1 is not a transcription factor but functions through its topoisomerase I-related motif as a part of the recombination machinery. No homology has been reported between RAG-2 and other genes. Characteriza- tion of the RAG-l and RAG-2 proteins has not been fruitful so far, mainly because of the difficulties in obtaining intact soluble protein products of these genes. Continued high expression of RAG-l and RAG-2 is toxic to cells; the toxicity could be due to the uncontrolled rearrangement of the genome of cells expressing the proteins (71). Bio- chemical characterization of these proteins should be done to under- stand their mode of action.

The gene mutated in scid mice is implicated as one component of the V(D) J recombinase (72). The scid mutation causes severe immuno- deficiency due to the virtual absence of both T and B cells. The V(D)J recombination is defective in scid lymphocytes. The defect is characterized by large deletions at the coding joints resulting in non-functional immunoglobulin genes (73, 74). In contrast, the formation of the signal joints remains intact in scid mutant. Non-lymphoid cells from scid mice

show increased sensitivity to radiation (75). The scid gene product is, therefore, a factor involved in general DNA repair.

Taccioli et al. analyzed V(D) J recombination in various CHO cell lines defective in DNA repair (76). By assaying the V(D)J recombination of plasmid recombination substrates after co-transfection of RAG- 1 and RAG-2 genes, two CHO cell lines, xrs-6 and XR-1, were found to have impaired V(D)J recombination. They are defective in forming both coding and signal joints and, therefore, are different from the scid mutation in which the signal joints are formed normally. The radiosen- sitive phenotypes of the xrs-6 and XR-1 cell lines could be corrected by transfection of different human chromosomes (chromosome 2 or 5, respectively), indicating that they are independent mutations. There are, therefore, at least three genes which are involved in V(D)J recombination as components of the general DNA repair machinery.

Many proteins involved in DNA repair also function in DNA replication processes. Recombination, repair, and replication of DNA apparently share a broad base of common mechanisms.

Over the past many years the structural changes of DNA induced

178 C. OK4 AND M. KAWAICHI

by V(D)J recombination have been almost completely described, yet the biochemical reaction mechanisms and regulatory processes of V(D)J recombination remain unknown. Isolation of factors in the two types of machineries in V(D) J recombination should lead us to complete understanding of this unique rearrangement of genomes.

SUMMARY

We have constructed transgenic mice carrying an artificial substrate of V(D)J recombination. In this substrate, the only DNA fragments derived from Ig genes were short stretches of recombination signal sequences. This artificial substrate was rearranged at high frequency in lymphocytes, although in non-lymphoid cells no rearrangement was detected even by a sensitive PCR assay. This result indicates that the V(D)J recombination requires only the signal sequences and that a recombination similar to the V(D)J recombination does not occur in non-lymphoid tissues including the central nervous tissue.

A protein binding to the V(D)J recombination signals was purified and its cDNA was cloned. This protein, termed RBP-JK, was initially considered to be involved in V(D)J recombination because of its DNA binding specificity and structural similarity to site-specific recombinases known as the integrase family. However, further study on the Drosophila homolog of RBP- JK indicated that RBP- JKprobably functions as a transcription factor in the differentiation of the peripheral nervous tissues. The exact function of RBP- JK is still unknown. Analogous to the Drosophila gene, it is suggested that mouse RBP-JK participates in the regulation of differentiation of various tissues.

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