Page 77 - Clinical Immunology_ Principles and Practice ( PDFDrive )

62 Part one Principles of Immune Response

Lymphoid-specific expression of RAG-1 and RAG-2 limits
V D J
V(D)J recombination to B and T lymphocytes. To ensure that
TCR genes are rearranged to completion only in T cells and Ig
genes are rearranged to completion only in B cells, V(D)J
RAG1, 2 recombination is further regulated by limiting the accessibility
of the appropriate gene segments to the specific lineage as well
as to the specific stage of development. For example, H chain
genes are typically assembled before L chain genes are.
V D J
The RAG-1 and RAG-2 recombinases cooperatively associate
with 12-bp and 23-bp RSSs and their flanking coding gene
segments to form a synaptic complex. Typically, the initial event
will be recognition of the nonamer sequence of a 12-bp spacer
RSS by RAG-1, which appears to function as the catalytic
V D
component of the recombinase. RAG-1 binding to the heptamer
provides specificity. RAG-2 does not bind DNA independently
J
but does make contact with the heptamer when in a synaptic
complex with RAG-1. Binding of a second RAG-1 and RAG-2
Nonhomologous end joining complex to the 23-bp, two-turn RSS permits the interaction of
the two synaptic complexes to form what is known as a paired
complex. Creation of this paired complex is facilitated by the
actions of the DNA-bending proteins HMGB1 and HMGB2 and
Signal joint by the presence of a divalent metal ion.
After paired complex assembly, the RAG proteins single-strand
cut the DNA at the heptamer sequence. The 3’ OH of the coding
+
sequence ligates to 5’ phosphate and creates a hairpin loop. The
V D J clean-cut ends of the signal sequences enable formation of precise
signal joints. However, the hairpin junction created at the coding
ends must be resolved by renicking the DNA, usually within 4–5
Coding joint
nucleotides from the end of the hairpin. This forms a 3’ overhang
that is amenable to further diversification. It can be filled in via
V D J DNA polymerases, nibbled back, or serve as a substrate for
TdT-catalyzed N addition. DNA polymerase µ, which shares
FIG 4.5 VDJ Recombination. Lymphoid-specific recombinase homology with TdT, appears to play a role in maintaining the
activating gene (RAG)-1 and RAG-2 bind to the recombination integrity of the terminus of the coding sequence.
signal sequences (RSSs) flanking V, D, or J gene segments, The cut ends of the coding sequence are then repaired by the
juxtapose the sequences, and introduce precise cuts adjacent NHEJ proteins. NHEJ proteins involved in V(D)J recombination
to the RSS. Components of the nonhomologous end joining include Ku70, Ku80, DNA-PKcs, Artemis, XRCC4, XLF (Cernun-
repair pathway subsequently unite the cut RSS to form a signal nos), and ligase 4.
joint, and the coding sequences of the rearranging gene segments Ku70 and Ku80 form a heterodimer (Ku) that directly associ-
to form a coding joint. ates with DNA double-strand breaks to protect the DNA ends
from degradation, permit juxtaposition of the ends to facilitate
coding end ligation, and help recruit other members of the repair
complex. The DNA protein kinase catalytic subunit (DNA-PKcs)
RSS (12-bp spacer) will preferentially recognize a two-turn signal phosphorylates Artemis, inducing an endonuclease activity
sequence (23-bp spacer). This helps prevent wasteful V-V or J-J that plays a role in the opening of the coding joint hairpin.
rearrangements. Thus absence of DNA-PKcs or Artemis inhibits proper coding
Initiation of the V(D)J recombination reaction requires joint formation. Signal joint formation is normal in Artemis
recombination activating genes 1 and 2 (RAG-1 and RAG-2). deficiency, but it is impaired in the absence of DNA-PKcs.
27
These genes are expressed only in developing lymphocytes. The Finally, XRCC4, XLF, and ligase 4 help rejoin the ends of the
gene products RAG-1 and RAG-2 act by precisely introducing a broken DNA.
DNA double-strand break (DSB) between the terminus of the Depending on the transcriptional orientation of the rear-
rearranging gene segment and its adjacent RSS (Fig. 4.5). These ranging gene segments, recombination can result in either
breaks are then repaired by ubiquitously expressed components inversion or deletion of the intervening DNA (see Fig. 4.3). The
of a DNA repair process that is known as nonhomologous end products of inversion remain in the DNA of the cell, whereas
joining (NHEJ). Lack-of-function mutations in NHEJ proteins deletion leads to the loss of the intervening DNA. The increased
yields susceptibility to DNA damage in all cells of the body and proximity of the V promoter to the C domain enhancers promotes
can lead to an SCID phenotype (Chapter 35). the subsequent transcription of the Ig gene product.
The NHEJ process creates precise joins between the RSS There is a price to the use of V(D)J recombination to create
ends and imprecise joins of the coding ends. TdT, which is antigen receptor diversity. Aberrant recombination in nonreceptor
expressed only in lymphocytes, adds non-germline–encoded genes can create deleterious genomic rearrangements that promote
26
nucleotides (N nucleotides) to the coding ends of the recombina- B-cell and T-cell neoplasias. For example, deletional recombina-
tion product. tion at the SIL/SCL locus and in Notch1, Izkf1, PTEN, and other