*2.2.8.1 Enzymatic mechanisms of recombination: RAG and RSS*

Rearrangements require two major steps: double strand-breaks (DSB) and repair of these breaks.

#### **Figure 3.**

*Cleavage site of RAG proteins at the V, D, and J gene segments of the IGH locus: RSS positions. RAG: recombination-activating gene, RSS: recombination sequence signal, IGH: immunoglobulin heavy chain locus.*

**27**

**Figure 4.**

*Immunogenetic Aspect of B-Cell Antigen Receptor Diversity Generation*

Both recombination-activating gene 1 (RAG-1) and RAG-2 recombinase enzymes, expressed exclusively in developing lymphocytes, are required to generate DSBs [6] at the level of recombinant signal sequences (consensus RSS, recombination sequence signal), flanking all functional V, D, and J gene segments, on the side that will be joined, *i.e.*, on the 3′ side of V segments, on both sides of D segments

*RSS motifs. There are two types of RSS, one includes a 12-nucleotide spacer (12-RSS), and the other includes a 23-nucleotide spacer (23-RSS). Both 12-RSS and 23-RSS include a highly conserved palindromic heptamer and* 

The RSS motifs are composed of a conserved palindromic heptamer and conserved nonamer motifs, separated by an intervening variable-sequence spacer of fixed length corresponding to 12 or 23 nucleotides (the resulting signals are referred to as 12-RSS or 23-RSS, respectively). From the architectural point of view of the H chains, all the V segments are tracked by a 23-RSS (on the 3′ side), the D segments are "framed" (on the 5′ and 3′ sides) by 12-RSS, and the J segments are preceded by 23-RSS (on the 5′ side). Regarding the L chain genes, the V segments are tracked by 23-RSS (on the 3′ side), and the J segments are preceded by 12-RSS (on the 5′ side) [8]. Only dissimilar RSS associations are efficiently recombined. Thus, each recombination that joins two gene segments occurs between 12-RSS and 23-RSS: this is known as the 12/23 rule. In the H chain recombination, the fact that the V and J segments are naturally both flanked by 23 nucleotide spacers (23-RSS), a connection between these two segments is not possible directly, but is done indirectly if they recombine with D elements, which are flanked on both sides by 12-RSS. After 12-RSS recombination with 23-RSS, the intermediate DNA will either be deleted or inverted depending on the orientation of the two signals (**Figure 4**). RAG-induced

DSBs are then resolved by nonhomologous end joining (NHEJ) pathway.

The assembly between the V, D, and J segments is done according to the sequential model as reported above. The V(D)J recombination mechanisms can be

*2.2.8.3 RAG action and NHEJ repair*

(3′, 5′), and on the 5′ side of J segments [7] (**Figure 3**).

*nonamer sequences. Bp: base pairs, RSS: recombination sequence signal.*

*2.2.8.2 Architecture of RAG-induced DSBs: the 12/23 rule*

*DOI: http://dx.doi.org/10.5772/intechopen.90637*

*Immunogenetic Aspect of B-Cell Antigen Receptor Diversity Generation DOI: http://dx.doi.org/10.5772/intechopen.90637*

#### **Figure 4.**

*Normal and Malignant B-cell*

random.

*repair*

of these breaks.

a potential repertoire of at least 107

and can therefore recognize up to two antigens.

*2.2.4 Rearrangements to a nonfunctional allele*

*2.2.6 D and J segments-CDR3 loop/IgH chain*

diversity. In total, molecular mechanisms of genetic recombination could result in

clone of such a cell repertoire contains only a few cells that are capable of recognizing only one antigen; exceptionally, a T-cell clone can express two different receptors

Two-thirds of rearrangements produce a nonfunctional allele for at least three

Some loci include only one V, D, or J gene segment. In these cases, all diversity is derived from junctional diversity or subsequent mechanisms of diversity, such as somatic mutation in IG loci, or from gene conversion in IG loci of some species.

D and J segments/genes encode amino acid sequences of the third loop of the immunoglobulin domain, which corresponds to the CDR3 region. If they had the

The transcription of the recombined IG gene gives rise to a functional messenger RNA, after elimination of introns, including any J segment/gene located between that which is joined to D and C segments. A similar process takes place in L chain loci.

Rearrangements require two major steps: double strand-breaks (DSB) and repair

*2.2.8 Molecular mechanisms of rearrangements: DNA double strand-breaks and* 

*Cleavage site of RAG proteins at the V, D, and J gene segments of the IGH locus: RSS positions. RAG: recombination-activating gene, RSS: recombination sequence signal, IGH: immunoglobulin heavy chain locus.*

main reasons: (i) the reading frame of V and C regions is correctly aligned in only one-third of the cases, (ii) the codons contain three-nucleotide, and (iii) the number of nucleotides inserted or eliminated in the junctions is essentially

*2.2.5 Diversity of receptors in the case of loci with a single V, D, or J segment*

same reading frame, recombination can give rise to an IgH chain.

*2.2.7 Transcription and generation of functional messenger RNA*

*2.2.8.1 Enzymatic mechanisms of recombination: RAG and RSS*

antigen-specific recognition sites/receptors. Each

**26**

**Figure 3.**

*RSS motifs. There are two types of RSS, one includes a 12-nucleotide spacer (12-RSS), and the other includes a 23-nucleotide spacer (23-RSS). Both 12-RSS and 23-RSS include a highly conserved palindromic heptamer and nonamer sequences. Bp: base pairs, RSS: recombination sequence signal.*

Both recombination-activating gene 1 (RAG-1) and RAG-2 recombinase enzymes, expressed exclusively in developing lymphocytes, are required to generate DSBs [6] at the level of recombinant signal sequences (consensus RSS, recombination sequence signal), flanking all functional V, D, and J gene segments, on the side that will be joined, *i.e.*, on the 3′ side of V segments, on both sides of D segments (3′, 5′), and on the 5′ side of J segments [7] (**Figure 3**).

#### *2.2.8.2 Architecture of RAG-induced DSBs: the 12/23 rule*

The RSS motifs are composed of a conserved palindromic heptamer and conserved nonamer motifs, separated by an intervening variable-sequence spacer of fixed length corresponding to 12 or 23 nucleotides (the resulting signals are referred to as 12-RSS or 23-RSS, respectively). From the architectural point of view of the H chains, all the V segments are tracked by a 23-RSS (on the 3′ side), the D segments are "framed" (on the 5′ and 3′ sides) by 12-RSS, and the J segments are preceded by 23-RSS (on the 5′ side). Regarding the L chain genes, the V segments are tracked by 23-RSS (on the 3′ side), and the J segments are preceded by 12-RSS (on the 5′ side) [8]. Only dissimilar RSS associations are efficiently recombined. Thus, each recombination that joins two gene segments occurs between 12-RSS and 23-RSS: this is known as the 12/23 rule. In the H chain recombination, the fact that the V and J segments are naturally both flanked by 23 nucleotide spacers (23-RSS), a connection between these two segments is not possible directly, but is done indirectly if they recombine with D elements, which are flanked on both sides by 12-RSS. After 12-RSS recombination with 23-RSS, the intermediate DNA will either be deleted or inverted depending on the orientation of the two signals (**Figure 4**). RAG-induced DSBs are then resolved by nonhomologous end joining (NHEJ) pathway.

#### *2.2.8.3 RAG action and NHEJ repair*

The assembly between the V, D, and J segments is done according to the sequential model as reported above. The V(D)J recombination mechanisms can be

#### *Normal and Malignant B-cell*

generated experimentally *in vitro* by mixing DNA (as an enzyme substrate) with the endonuclease RAG-1 and RAG-2 proteins.

The kinetics of main rearrangement events are described according to the following steps (**Figure 5**):


**29**

**Figure 5.**

disappears from the cell [16–18].

*Immunogenetic Aspect of B-Cell Antigen Receptor Diversity Generation*

bordering the RSS heptamer. When the two gene segments that are joined have the same orientation, which is observed in most cases, the signal joint is excised on a circular DNA segment and generates a circular episome (an extrachromosomal circular DNA) located between the two coding regions, which later

*Molecular mechanisms of V(D)J recombination and junctional diversity generation (adapted from [3]). (A) Main steps of V(D)J recombination. (B) Edge modification of coding regions and junctional diversity. DNA-PKcs: DNAdependent serine/threonine protein kinase complex, DSB: double strand-breaks, N: nongermline/nontemplated nucleotide, P: short complementary palindromic sequences, XRCC4: X-ray cross-complementing gene 4.*

*DOI: http://dx.doi.org/10.5772/intechopen.90637*

*Immunogenetic Aspect of B-Cell Antigen Receptor Diversity Generation DOI: http://dx.doi.org/10.5772/intechopen.90637*

#### **Figure 5.**

*Normal and Malignant B-cell*

lowing steps (**Figure 5**):

endonuclease RAG-1 and RAG-2 proteins.

generated experimentally *in vitro* by mixing DNA (as an enzyme substrate) with the

The kinetics of main rearrangement events are described according to the fol-

a.*Formation of a synapse***.** RAG forms synaptic complexes, only with one 12-RSS and one 23-RSS, according to a 12/23 dogma that governs the recombination

b.*RAG and RSS binding*. A key role is attributed to RAG-1 in DNA binding and catalysis, as well as in interactions with RAG-2, high-mobility group box 1 and 2 (HMG1/2), and itself, given its structure that contains a nonamer binding domain (NBD) required for stable recruitment of RAG proteins into RSS [10]. So, RAG-1 recruit RAG-2 after binding of an RSS and then RAG-2 maintains

c.*DSB generation*. The two RAG-RSS complex will generate DSB at the level of the two RSS (between a gene segment and an RSS), by first a DNA strand cleavage, between the 5′ end of the RSS heptamer and the region encoding the antigen receptor and then the other DNA strand, after reaction of the free 3′-hydroxyl

sequences are covalently bound, thereby forming hairpin structures, and will

e.*Hairpin opening-DNA synthesis and planning of extremities*. The hairpin ends are opened and rendered straight by DNA synthesis or nucleolytic cleavage planning using a nuclease, which would appear to be "Artemis." The hairpin cleavage reaction can be done in two points: it can either be cleaved in the middle or a few nucleotides on one side of the center, producing offset cuts.

f. *Formation of N regions*. During IGH V-IGH D and IGH D-IGH J joining [2], TdT, an important enzyme required for the junctional diversity generation, can intervene, before the juxtaposition of the two coding regions, to generate a higher level of diversity (see above), by catalyzing the elimination of nucleotides and/or the addition of nontemplated nucleotides to the 3′ ends, which will produce the so-called N regions (nongermline/nontemplated nucleotide) [11]. So N1 and N2 regions are created between the V and D genes and between the

g.*Deletion/insertion of P nucleotides*. Deletions or insertions of short complementary palindromic sequences, so-called P nucleotides, are generated through endonuclease activity and repair around the asymmetric opening of hairpin loops that form at the ends of the gene segments to be joined as part of the rearrangement process and produce short, self-complementary single stranded extensions that can be incorporated into junctions, or may alternatively be

h.*NHEJ ligation*. The nonhomologous DNA ends of the two coding regions are repaired/ligated using an NHEJ repair system, generating, on the one hand, coding joints, in which the gene segments are joined [14, 15], and, on the other hand, RSS-containing signal joints, resulting from the direct joining of the DSB

removed *via* exonuclease activity [12] (for review, see [13]).

fidelity, and then the rearrangements are started [9].

the RAG-1-RSS complex and binds to a second RSS.

(3'-OH) group with a phosphodiester bond of this strand.

be maintained within synaptic complexes.

D and JH genes, respectively.

d.*Formation of hairpin structures*. Nucleotides of the cleaved-end coding

**28**

*Molecular mechanisms of V(D)J recombination and junctional diversity generation (adapted from [3]). (A) Main steps of V(D)J recombination. (B) Edge modification of coding regions and junctional diversity. DNA-PKcs: DNAdependent serine/threonine protein kinase complex, DSB: double strand-breaks, N: nongermline/nontemplated nucleotide, P: short complementary palindromic sequences, XRCC4: X-ray cross-complementing gene 4.*

bordering the RSS heptamer. When the two gene segments that are joined have the same orientation, which is observed in most cases, the signal joint is excised on a circular DNA segment and generates a circular episome (an extrachromosomal circular DNA) located between the two coding regions, which later disappears from the cell [16–18].

The junction system comprises a number of ubiquitous repair proteins (present in both B-cells and T-cells), which would allow rearrangement of IG genes in B-cell precursors, but rarely in T-cell precursors, and TCR gene rearrangements in T-cell precursors, but rarely in B-cell precursors. It includes in particular the catalytic subunit of a nuclear DNA-dependent serine/threonine protein kinase complex (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related (PIKK) family of protein kinases, composed of a heterodimer of Ku proteins that bind free DNA ends given their strong affinity (Ku70/Ku80 [encoded, respectively, by X-ray cross-complementing gene 5 (XRCC5) and XRCC6 genes in humans, and also called Ku86]) [19], XRCC4, DNA ligase IV and Artemis [3]. TdT is also recruited into the junction system and is involved in the formation of the coding joints, alongside Artemis and DNA-PKcs. Nevertheless, TdT is only rarely recruited into rearrangements that occur during fetal life, so that junctional diversity is limited.
