Reason behind antibody diversity and mechanism of V(D)J recombination by Sourin Adhikary.

1. Antibody diversity with special emphasis on V(D)J recombination
Submitted by: Sourin Adhikary
Roll No. : 100190232002013
Reg. No. : KNU19002619
M.Sc. 4th sem, Department of Animal Science
Kazi Nazrul University

2. INTRODUCTION
Antibody are antigen binding proteins present on the B-cell membrane and secreted by
plasma cell.
Antibody diversity:
 There are millions of antigen/epitope.
 Our immune system has the ability to produce specific antibody (variable region) against
all antigen.
 This diversification in antibody production is known as antibody diversity.
V(D)J recombination:
 V(D)J recombination is the mechanism of DNA rearrangement that occurs only in
developing lymphocytes during the early stages of B and T cell maturation. It results in the
highly diverse repertoire of antibodies/immunoglobulins and T cell receptors found in B
cells and T cells, respectively.
 The germline genes cannot be transcribed into mRNA directly that encode functional
antigen receptor proteins.
 Functional antigen receptor genes are created only in developing B and T lymphocytes
after DNA rearrangement brings randomly chosen V, (D), and J gene segments.
 In each lymphocyte to form a single V(D)J exon that will code for the variable region of
an antigen receptor protein.
But

3. PROBLEM
Out of 40000 genes in our genome only few gene codes for Ig. But our immune system
apparently produce antibody in the order of 107 or more . So how is an infinite diversity of
specificity generated from finite amounts of DNA?

4. 1. Dreyer & Bennett experiment (1965)
According to Dreyer & Bennett postulates, for a single isotype of antibody there may be:
 A single C region gene encoded in the germ line and separate from the V region genes.
 Multiple choices of V region genes available.
 A mechanism to rearrange V and C genes in the genome so that they can fuse to form a
complete immunoglobulin gene.

5. 2. Tonegawa’s experiment
These experiment showed that the antibody light chain was encoded in the germ line by not
one but three families of gene segments separated by kilobases of DNA.
Fig: The antibody light-chain gene encodes three families of DNA segments

6. 3. Lee Hood’s experiment
According to Hood & his colleagues, the heavy chain (H-chain) encoded gene contains three
exon (V, D and J). There is no D region in light chain.
Fig: Heavy chain V region gene segments in embryo (germline DNA)
So, I think now the concept of V, D and J are clear to all

7. Generation of antibody diversity
1. Combinatorial V(D)J joining:
There can random joining of any V gene with any J gene that produce L-chain. Similarly any
V gene can join any D or J gene to produce H-chain.
Using functional V, D, and J genes:
40 VH x 27 DH x 6JH = 6480 combinations
D can be read in 3 frames: 6480 x 3 = 19,440 combinations
29 Vκ x 5 Jκ = 145 combinations
30 Vλ x 4 Jλ = 120 combinations
Total 265 different light chains
If H and L chains pair randomly as H2L2 i.e. 19,440 x 265 = 5,151600 possibilities due only
to COMBINATORIAL diversity.

8. 2. Junctional and insertional diversity:
During the V(D)J joining to produce on antibody, a variable number of nucleotides are often
deleted from the ends of joining genes and other randomly chosen nucleotides are added . This
random loss and gain of nucleotides is called junctional/insertional diversification.

9. 3. Somatic hypermutation:
 The variable region of germline DNA are very prone to a high rate of somatic mutation
during B-cell development. It is called as somatic hypermutation and it occurs 10,000 times
higher than normal mutation rate.
 Occur within germinal centers of secondary lymphoid organ after exposure to an antigen.
 Individual nucleotide in V(D)J units are replaced with alternative nucleotide.
 It potentially alter the specificity of encoded Ig.
 Following exposure to an antigen, B cells with higher affinity receptors selected for
survival.
 Such B-cells undergo affinity maturation takes place in germinal centers.
* Affinity maturation is the process whereby the immune system generates antibodies of
higher affinities during a response to antigen.
Wu-Kabat analysis compares point
mutations in Ig of different
specificity.
Somatic hypermutation

10. V(D)J RECOMBINATION

11. OVERVIEW
 For light chain, any of Vλ gene can combine with any of Jλ-Cλ combination (same in κ
chain also).
 For heavy chain, any of VH gene can combine with any of DH – JH – CH combination.
 Single antigen specific immunocompetent cell is produced.
Fig: Overview of
recombination of
immunoglobulin
variable region genes

12. Recombination Signal Sequence (RSS)
Recombination is catalyzed by a set of enzymes and is directed to the appropriate sites on
the Ig gene by recognition of specific DNA sequence motifs called RSSs. These sequence
ensure that one of each type of segment (V and J for the light chain, or V, D, and J for the
heavy chain) is included in the recombined variable region gene.
Fig: Two conserved
sequences in light-chain and
heavy-chain DNA function as
recombination signal
sequences (RSSs). (a) Both
signal sequences consist of a
conserved heptamer and conserved
AT-rich nonamer; these are
separated by nonconserved spacers
of 12 or 23 bp. (b) The two types of
RSS have characteristic locations
within -chain, -chain, and
heavychain germ-line DNA. During
DNA rearrangement, gene segments
adjacent to the 12-bp RSS can join
only with segments adjacent to the
23-bp RSS

13. Molecular explanation of the 12-23 rule
Continue……
23-mer = two turns 12-mer = one turn
7 J
D
7
7
V 9
9
23 12
Intervening DNA
of any length

14. Molecular explanation of the 12-23 rule
V1 V2 V3 V4
V5
V6
V7
V8
V9 D J
12-mer
23-mer
V1
7
7 D J
9
9
V2
V3
V4
V5
V6
V7
V8
V9
Loop of
intervening DNA
is excised
• Heptamers and nonamers align
back-to-back
• The shape generated by the RSS’s
acts as a target for recombinases
• An appropriate shape can not be formed if two 23-mer flanked elements attempted to join
(i.e. the 12-23 rule)

15. PROBLEM
 How do V region find J regions and why don’t they join to C region?
 How does DNA break and rejoin?

16. 12-23 rule – A gene segment flanked by a 23mer RSS can only be linked to a segment
flanked by a 12mer RSS.
Recombination Signal Sequence (RSS)
So, I think the answer of the previous first question is now clear to all
HEPTAMER - Always contiguous with
coding sequence
12 7 D 7 12 9
9
NONAMER - Separated from the heptamer by a
12 or 23 nucleotide spacer
VH 7 23 9 9 23 7 JH
9 12 7 D 7 12 9
VH
7 23 9 9 7
23 JH

17. Mechanism of V(D)J recombination
Step 1:
Recognition of the heptamer-nonamer Recombination Signal Sequence (RSS) by the
RAG1/RAG2 enzyme complex. The RAG1/2 recombinase forms a complex with the
heptamer-nonamer RSSs contiguous with the two gene segments to be joined. Complex
formation is initiated by recognition of the nonamer RSS sequences by RAG1 and the 12-
23 rule is followed during this binding.
Fig: RAG1/2 and HMG proteins
bind to the RSS and catalyze
synapse formation between a
V and a J gene segment.

18. Step 2:
One-strand cleavage at the junction of the coding and signal sequences. The RAG1/2
proteins then perform one of their unique functions: the creation of a single-strand nick, 5’ of
the heptameric signal sequence on the coding strand of each V segment and a similar nick on
the non-coding strand exactly at the heptamer-J region junction.
Fig: RAG1/2 performs a single
stranded nick at the exact 5’ border
of the heptameric RSSs bordering
both the V and the J segments.

19. Step 3:
Formation of V and J region hairpins and blunt signal ends. The free 3’ hydroxyl group at the
end of the coding strand of the VK segment now attacks the phosphate group on the opposite,
non-coding VK strand, forming a new covalent bond across the double helix and yielding a
DNA hairpin structure on the V-segment side of the break (coding end). Simultaneously, a
blunt DNA end is formed at the edge of the heptameric signal sequence. The same process
occurs simultaneously on the J side of the incipient joint. At this stage, the RAG1/2 proteins
and HMG proteins are still associated with the coding and signal ends of both the V and J
segments in a post cleavage complex.
Coding end Signal end
Fig: The hydroxyl group attacks the
phosphate group on the non-coding
strand of the V segment to yield a
covalently-sealed hairpin coding end
and a blunt signal end.

20. Step 4:
Ligation of the signal ends. DNA ligase IV then ligates the free blunt ends to form the signal
joint.
Fig: Ligation of the signal ends

21. Step 5:
Hairpin cleavage. The hairpins at the ends of the V and J regions are now opened in one of
three ways. The identical bond that was formed by the reaction described in step 3 above,
may be reopened to create a blunt end at the coding joint. Alternatively, the hairpin may be
opened asymmetrically on the “top” or on the “bottom” strand, to yield a 5’ or a 3’ overhang,
respectively. A 3’ overhang is more common in in vivo experiments. Hairpin opening is
catalyzed by Artemis, a member of the NHEJ pathway.
Fig: Opening of the hairpin
can result in a 5’ overhang, a
3’ overhang, or a blunt end.
Here 3’ overhang has been
showed

22. Fig: Cleavage of the
hairpin generates sites
for P nucleotide
addition.
Step 6:
Overhang extension, leading to palindromic nucleotides. In Ig light-chain rearrangements,
the resulting overhangs can act as substrates for extension DNA repair enzymes, leading to
double stranded palindromic (P) nucleotides at the coding joint. P nucleotide addition can
also occur at both the VD and DJ joints of the heavy-chain gene segments, but, as described
below, other processes can intervene to add further diversity at the VH-D and D-JH
junctions.

23. Step 7:
Ligation of light-chain V and J Segments. Members of the non-homologous end joining
(NHEJ) pathway repair both the signal and the coding joints, but the precise roles of each,
and potentially other enzymes in this process, have yet to be fully characterized.
Fig: Ligation of light chain
V and J regions by ku70/80
complex, artemis, DNA-
PKcs, XRCC4.

24. Step 8:
Exonuclease trimming. An exonuclease activity, which has yet to be identified, trims back
the edges of the V region DNA joints. Since the RAG proteins themselves can trim DNA
near a 3 fl ap, it is possible that the RAG proteins may cut off some of the lost nucleotides.
Alternatively, Artemis has also been shown to have exonuclease, as well as endonuclease
activity, and could be the enzyme responsible for the V(D)J-associated exonuclease function.
Fig: In heavy chain VD and DJ
joints only: Exonuclease
cleavage results in loss of coding
nucleotides at joint – can occur
on either or both sides of joint

25. Step 9:
N nucleotide addition and Ligation and repair of the heavy-chain gene. Non-templated (N)
nucleotides are added by terminal deoxynucleotidyl transferase (TdT) to the coding joints of
heavy chain genes after hairpin cleavage. This enzyme can add up to 20 nucleotides to each
side of the joint. The two ends are held together throughout this process by the enzyme
complex, and again, loss of the correct phase may occur if nucleotides are not added in the
correct multiples of three required to preserve the reading frame.
Fig: Non-templated nucleotides
(in red) are added to the coding
joint by TdT. Ligation of heavy
chain by DNA ligase IV and
NHEJ proteins.

26. For more understanding and reading……
 Text book of Immunology, KUBY, 7th edition, chapter 5, Organisation and expression of
Immunoglobulin gene

27. Thank you all