V (D) J RECOMBINATION

Antibody Diversity: V-D-J gene segment recombination in Immunoglobulin Heavy Chains V-J gene segment recombination in Immunoglobulin Light Chains

Sources of Antibody Diversity: V-D-J gene segment recombination in Immunoglobulin Heavy Chains V-J gene segment recombination in Immunoglobulin Light Chains

Antibody Diversity
Human cells do not have enough DNA to have separate

genes for each antibody molecule. Different segments of antibody genes can be shuffled to form different antibodies.

Regions of antibody genes: V- variable D Diversity J - Joining

Immunoglobulin Structure
Lambda & Kappa Light Chains - small polypeptide subunit of an antibody. Two types of light chains: kappa () chain, encoded by the immunoglobulin kappa locus (IGK) on chromosome 2 lambda () chain, encoded by the immunoglobulin lambda locus (IGL) on chromosome 22

Once genetically determined, a specific B lymphocyte expresses only one class of light chain that remains fixed for the life of the B lymphocyte. Glycosylation of heavy chains produces the IG glycoprotein

Immunoglobulin Structure
Only one type of light chain is present in a typical

antibody, thus the two light chains of an individual antibody are identical. Each light chain is composed of two tandem immunoglobulin domains (CL and CV) The approximate length of a light chain protein is from 211 to 217 amino acids.

Light Chain Domains:

one constant (CL) domain one variable domain (VL) that is important for binding antigen

Immunoglobulin Heavy Chains

Immunoglobulin Heavy Chain (IgH) is the large polypeptide subunit of an antibody different types of heavy chain exist that define the class or isotype of an antibody. All heavy chains contain a series of immunoglobulin domains: One variable domain (VH) that is important for binding antigen Several constant domains (CH1, CH2, etc.).

Immunoglobulin Classes
Five types of immunoglobulin heavy chains:

, , , and . These define classes IgG, IgD ,IgA, IgM and IgE. Heavy chains and have approximately 450 amino acids. Heavy chains and have approximately 550 amino acids.

Heavy Chain Regions

heavy chains have two regions:

a constant region (which is the same for all

immunoglobulins of the same class but differs between classes).

Heavy chains , and have a constant region composed of three

tandem (in a line next to each other) immunoglobulin domains but also have a hinge region for added flexibility. Heavy chains and have a constant region composed of four domains.

a variable region that differs between different B cells, but

is the same for all immunoglobulins produced by the same B cell or B cell clone. The variable domain of any heavy chain is composed of a single immunoglobulin domain. These domains are about 110 amino acids long.

 Three gene segments of diversity

Two in the Lambda & Kappa Light Chain
a) V- variable
b) J- joining

Three in each Heavy Chain

a) b)

c)

V- variable D- diversity J- joining

Conserved gene segments

a) L- leader- In front of every V. i. Codes for 19 amino acids ii. Makes up hydrophobic segment

b) C- constant- determines Ig isotype

c)

M- membrane exons
i. ii. Only in heavy chains Constitutes the cytoplasmic (CY) exon & the transmembrane (TM) exon

Gene Organization
Antibody molecules and B cell receptors are composed of heavy and light chains with both constant (C) and variable (V) regions that are encoded by genes on three loci. Immunoglobulin heavy locus (IGH) on chromosome 14, containing gene segments for the immunoglobulin heavy chain Immunoglobulin kappa () locus (IGK) on chromosome 2, containing gene segments for the immunoglobulin light chain Immunoglobulin lambda () locus (IGL) on chromosome 22, containing gene segments for the immunoglobulin light chain

 Recombination Signal Sequences (RSS) Located at:

3 end of V segment Bordering the D segment 5 end of the J segment

Separating Sequences
12- one turn 23- two turn

Palindromes
Heptamer

3 end of V and 5 side of J

Nonamer
3 end of J and 5 side of V

 Recombination Activation Genes (RAG) Encode for RAG Proteins Adjacent to each other Induce Ig gene recombination Expressed during G0 and G1 phases

Recombination between the variable (V), diversity (D) and joining (J) segments of antigen-receptor genes is catalyzed by the RAG protein complex (green; only D and J are shown here, blue). RAG targets complementary recombination signal sequences, 12RSS (orange) and 23-RSS (red). These sequences and the intervening DNA are cut out and join to form a DNA circle. The remaining coding sequences are also joined.

b: RAG-mediated translocations between chromosomes. RAG can mediate translocations by catalyzing V(D)J recombination involving the recombination signal sequence of an antigen-receptor locus and a complementary, but cryptic, recombination signal sequence adjacent to an oncogene.

Such translocations can occur through several related pathways.

c: Coding or recombination signal sequence ends may escape from chromosomal RAG post-cleavage complexes then, fuse with double-stranded breaks at other locations (translocation targets), sometimes near oncogenes.

d, Coding or recombination signal sequence ends may escape from chromosomal RAG post-cleavage complexes and fuse with RAGinitiated lesions induced at the Bcl-2 major breakpoint region (Mbr). The RAG-mediated nicks in the Mbr are hypothesized to arise as a result of the unusual DNA structures.

 RAG 1 & RAG 2 Endonucleases Responsible for recombination Ability to promote transcription depends on:
Accessibility to DNA B-cell differentiation transcription factor expression EBF and E box proteins E2A Initiate heavy chain Ig gene rearrangements

Bind to site-specific substrates (RSSs)

Upon binding to RSS they bind to each other Segments line up with one another Precise cut at heptamer-gene segment Cleaves DNA strand which ultimately creates hairpin loop Creates coding ends and signal ends

 RAG complex bound to DNA Nicks are induced Promotes binding of repair proteins
KU70 &KU80 Identify breaks KU80 recruits DNA-PK DNA-dependent protein kinase Encoded by PRKDC gene Part of Phosphotidylinositol-3-kinase, related kinase family
enzymes involved in cellular functions such as cell growth, proliferation,

differentiation, motility, survival and intracellular trafficking, which in turn are all involved in cancer
Binds to each broken DNA end
Recruit and activate other enzymes

 Artemis Protein artemis is a protein that in humans is encoded by

the DCLRE1C (DNAcross-link repair 1C) gene.

Encoded by the DCLRE1C (DNA cross-link repair 1C) gene
The protein has single-strand-specific 5'-3' exonuclease activity
When complexed with and activated by DNA-PK it displays

endonuclease activity on 5' and 3' overhangs and hairpins Opens coding-end hairpin loops

XRCC4 Encoded by XRCC4 gene Functions together with DNA ligase IV Repairs DNA double-strand breaks
Ligase: complexes with DNA repair protein XRCC1 to aid in sealing DNA during the process of nucleotide excision repair and recombinant fragments.

Ligase: complexes with DNA repair protein XRCC1 to aid in sealing DNA during the process of nucleotide excision repair and recombinant fragments.

 DNA ligase IV & XRCC4 Responsible for last steps of NHEJ Ligates gene segments together Ligates to form coding joint
Also forms signal joint

Junctional flexibility occurs P-nucleotide addition Result of Artemis and ligase activity

 Terminal deoxynucleotidyl Transferase (TdT) Type of DNA polymerase special to immature lymphocytes Catalyzes the addition of nucleotides to the 3' terminus of a DNA molecule
No template required 3 Overhang or blunt end repair

This is N region insertion

Product of recombination

 Allelic Exclusion Expression of a maternal/paternal gene over the otherPhenomenon of expressing one gene while silencing the other allele. B-cells make two choices
1. 2.

Express or light chains Use maternal or paternal genes

Regulated by sequence of events

Reducing RAG1 & RAG2 expression Degrades proteins (RAG2) Reduce recombinase access to heavy chain locus

 Regulation leads to: Germline configuration Successful rearrangements leading to suppression

Ability of allele to undergo recombination is controlled

through targeted changes in chromatin accessibility

Histone acetylation DNA methylation

 Begins with heavy chain expression Both Hc alleles accessible to recombinases Leads to efficient bi-allelic Dh-Jh rearrangement Productive rearrangement on one allele leads to:
Inhibition of Hc rearrangement on other allele Stimulation of -chain rearrangement

Nonproductive rearrangement leads to:

Rearrangement on second allelic chromosome If also nonproductive cell-death occurs

 Light chain isotype exclusion Begins with light chain rearrangement

If rearrangement fails it can be repeated with unused V-J segments up

to five times
light chain rearrangement begins if no light chain rearrangement

succeeds
Cycle repeats for this light chain as for the other

After successful heavy and light chain rearrangements,

further DNA rearrangements are prevented

Developing lymphocyte senses successful gene rearrangements

 Activation of transcription Cis-regulators

Promoters RNA binds here to begin transcription

Enhancers Enhance ability for transcription to occur

Silencers Operates in both directions over a distance to down regulate transcription

Trans-regulators
Transcription/Nuclear factors Provide link between external signals and gene expression

 Promoters Not strong enough itself to start transcription Located approximately 250kb away from enhancer
Assures no transcription of unrearranged gene Assures rearranged genes are influenced by same enhancer 5 of the C Will continue to work after class switching

Promoter and enhancer brought within 2kb after rearrangement

Must have trans-regulators to transcribe

 Once gene is rearranged and everything is in place: Transcription occurs mRNA is produced mRNA processing and post-transcriptional modifications Mature mRNA is translated Leader sequence is cleaved in a post-translational modification Newly formed Ig chain is formed

 Severe Combined Immunodeficiency (SCID) Omenn Syndrome

Due to mutation in RAG 1&2 genes

Chronic diarrhea Ear infections Babies die within a year if untreated