Much of the diversity in both TCR and Ig proteins is generated by
the combinatorial recombination at the DNA level of inherited gene
segments. Both TCR and Ig function as heterodimers, and each polypeptide
is comprised of a variable region [assembled by V(D)J recombination] and
a constant region (joined to the variable region by conventional
mRNA splicing). The variable portion of one polypeptide in each heterodimer
(Ig heavy chain and TCR β or δ chains)
is assembled from three elements named ‘V’, ‘D’ and ‘J’ segments.
The partner polypeptide (one of the Ig light chains κ or λ, or TCR α or γ) is assembled from V and J segments
only. In mice and humans these segments are usually inherited as
tandem clusters of related elements (Fig. A).
The organization has evolved and is quite different among phylogenetically
older vertebrates such as sharks. A detailed discussion of the origin of
this recombination system and the evolution of the chromosomal organization
of the various rearranging loci has recently appeared (9
and references therein, 10
Figure 1 1 The V(D)J
recombination reaction scheme. (A) The colored
bars represent the tandemly-arranged clusters of coding DNA segments
at the Ig heavy chain locus of the mouse. There are actually several
hundred V segments, many of which are pseudogenes. (more ...)
A brief discussion of the reaction steps in V(D)J recombination is
necessary to introduce the key concepts, but these have been reviewed
extensively. The reader is referred elsewhere for a comprehensive
introduction to the process from the perspective of the substrate
loci and the recombinant products (11
). The key terminology, elaborated in
Figure , is that the various ‘coding’ segments
(drawn as colored vertical bars) are physically cut and joined together.
The cuts occur in two steps, first by nicking a particular DNA strand
adjacent to the heptamer, followed by cleavage of the second strand
to yield a terminal hairpin on the coding DNA. The ligation of two coding
segments forms a ‘coding joint’, which is deliberately imprecise.
Small deletions, random insertions and occasional small palindromic
additions are common at these junctions and arise through the processing
of the intermediate hairpin end shown in Figure C.
The junctional diversity greatly increases the yield of different
receptor molecules, which otherwise would be limited to the multiplicative
product of the number of segments. The DNA ligation step is dependent
on proteins already known to participate in the repair of other
DNA double-strand breaks, the so-called NHEJ pathway (reviewed in 13
The cleavage is directed to a precise location by the presence
of recombination signal sequences (RSSs). These are represented by triangles
in Figure . The RSS is composed of conserved heptamer
and nonamer elements separated by an intervening spacer of fixed
length. The consensus sequence is shown in Figure D, but variations are frequent, and deviation
from an optimal sequence may modulate the efficiency with which particular
sites are used (20
through the intervention of additional protein factors (21
). The RSS elements have evolved over
). There are two classes
of RSS, which are distinguished by the length of their spacer regions.
Spacer length centers on 12 or 23 nt and the resulting signals are referred
to as 12-RSS or 23-RSS (Fig. , white and
black triangles, respectively). The existence of the two classes
helps solve a potential pitfall in the recombination system. While
it is desirable to allow recombination between, for example, any one
V segment and any one D segment, it would serve no useful purpose
to recombine two V segments with each other. This is avoided by
organizing the RSS elements such that only one length class is used
within a cluster of segments. In the case of the mouse Ig heavy
chain locus, each of the V regions is associated with 23-RSS elements.
The D regions in this locus are flanked on both sides by 12-RSS
elements, thus allowing recombination to occur both upstream and
downstream. Finally, the J segments of this locus utilize purely
23-RSS signals (Fig. B). The mechanism
of recombination incorporates a ‘12/23 rule’ (23
), which specifies that recombination
be permitted only between segments of complementary RSS length.
This allows V to D and D to J recombination but neatly prevents
the undesired occurrences. D segments contribute only short lengths
of coding information to the assembled polypeptide, typically from
two to nine amino acids. True devotees will appreciate the totally
different configuration of RSS elements at the mouse TCR δ locus.
The D elements there, in contrast, are flanked by one 12-RSS and
one 23-RSS, permitting D to D recombination and resulting in both
VDJ and VDDJ products. Both result in functional molecules [see
), figure 1].
The molecular implementation of the 12/23 rule is not fully
determined, but will be addressed later.
While the joining of coding sequences, as portrayed in Figure C, creates the assembled receptor gene, it
is important for several reasons that the DNA cleaved from the coding sequence,
which carries the RSS elements, also be rejoined. This DNA is shown
as the circular product in Figure C. The cleavage
reaction leaves blunt double-strand breaks at the border of the
and these are typically joined to each other in precise heptamer
to heptamer juxtaposition. These so-called ‘signal joints’ also
use the NHEJ pathway, but are distinguishable from coding joints.
Signal joints only rarely lose or gain bases and do not require
the protein DNA-PKcs (26
) for their formation. This probably
reflects the additional processing demanded by the coding ends.
Forming signal joints serves two apparent purposes. First, it prevents
the undesirable integration of these DNA ends into other locations as
studied in vitro
), and indirectly, in vivo
The second need for signal joint formation reflects other topologies of
the reaction. In the typical rendering of the reaction, as shown
in Figure C, the opposing orientation of
the RSS elements creates a deletion circle. This is the most common configuration
in natural loci. However, functional recombination does occur among
gene segments in which the participating RSS elements are in direct
orientation. Under this circumstance, rearrangement demands inversion
of the DNA between the RSS elements rather than deletion, and signal
joints are required for chromosomal integrity.