Immunoglobulin Gene Rearrangement Eliza Thapa Huizhi Zhao Jingwen Song

General Outline Introduction to immunoglobulin and V(D)J recombination Mechanism of V(D)J recombination Effect of V(D)J recombination

Immunoglobulin (Ig) http://factfile.org/wp-content/uploads/2015/03/Antibodies-Image.jpg serum glycoprotein produced by B cells against foreign molecules foreign molecules- antigen (bacteria, virus, pollens etc) antibody binds to specific antigen occurs in two forms- soluble form - Membrane bound form (BCR)

Structure of Immunoglobulin 2 heavy chains (H) 2 light chains (L) H and L linked by disulfide bonds Two (H-L) chain linked by disulfide bond amino terminal variable (V) region Carboxyl- terminal constant (C) region www.namrata.co/wp-content/uploads/2012/04/immunoglobulins-structure.jpeg

Types of Immunoglobulin Five classes of immunoglobulins Immunoglobulin D (δ) Immunoglobulin E (ε) Immunoglobulin G (γ) Immunoglobulin M (μ) Immunoglobulin A (α) Not found in humans Immunoglobulin Y Immunoglobulin W http://www.nature.com/nrm/journal/v3/n12/box/nrm972_BX1.html

Means of Ig Diversity Multiple germ-line gene segments Combinatorial V-(D)-J joining Junctional flexibility P-region nucleotide addition N-region nucleotide addition Somatic hypermutation Combinatorial association of light and heavy chains

Combinatorial V-(D)-J joining http://www.microbiologybook.org/mayer/gen-1.jpg

V(D)J recombination occurs during B-Cell Development

Light chain Gene rearrangement

Heavy chain gene rearrangement

How much variation is possible through recombining gene fragments? Over 1.5*107combinations of variable, diversity and joining gene segments are possible. Imprecise recombination and mutation increase the variability into billions of possible combinations

Mechanism of Rearrangement Recombination Signal Sequences (RSSs). Recombination Activating Genes: RAG-1 and RAG-2. The RAGs encode enzymes that play an important role in the rearrangement and recombination of the genes of immunoglobulin and T cell receptor molecules during the process ofVDJ recombination

Recombination Signal Sequence A short DNA sequence indicate the sites of recombination seven conserved nucleotides (a heptamer) that reside next to the gene encoding sequence a spacer (containing either 12 or 23 unconserved nucleotides) a conserved nonamer (9 base pairs) Nature Reviews Immunology 11, 251-263 (April 2011)

Recombination Signal Sequence Nature Reviews Immunology 11, 251-263 (April 2011) | doi:10.1038/nri2941 Nature Reviews Immunology 11, 251-263 (April 2011) | doi:10.1038/nri2941 Recombination Signal Sequence Two types of RSS exist Each V, D, or J gene segment is flanked by RSS The RSSs are present on the 3’ side (downstream) of a V region and the 5’ side (upstream) of the J region RSSs are recognized by a group of enzymes known collectively as the VDJ recombinase. RSSs are composed of seven conserved nucleotides (a heptamer) that reside next to the gene encoding sequence followed by a spacer (containing either 12 or 23 unconserved nucleotides) followed by a conserved nonamer (9 base pairs).  spacer of 12 nucleotides will be recombined with one that has a spacer containing 23 nucleotides). This is known as the 12/23 rule of recombination (or the one-turn/two-turn rule) http://biosiva.50webs.org/immunediversity.htm

Recombination Activating Gene RAG enzymes work as a multi-subunit complex to induce cleavage of a single double stranded DNA (dsDNA) molecule between the antigen receptor coding segment and a flanking recombination signal sequence (RSS). Schatz, David G., and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation." Annual review of genetics 45 (2011): 167-202.

V(D)J recombination: a complex containing RAG1 and RAG2 binds one RSS. This RAG-RSS complex then captures the second RSS (of the gene segment to be joined) in a process known as synapsis.  Cleavage by RAG1/2 occurs between the RSS heptamer and flanking coding sequence, and proceeds in two steps. A nick is made at the 5’ end of the RSS heptamer. The second step is a hairpinning step joining the 3’-hydroxyl to the phosphoryl group at the same nucleotide position on the other strand.  DNA cleavage is completed within the synaptic complex. The product of this first phase of V(D)J recombination is the “cleaved signal complex,” which contains four DNA ends: two blunt 5’-phosphorylated signal ends, and two coding ends terminating in DNA hairpin structures. Nature Reviews Immunology 8, 302-312 (April 2008)

V(D)J recombination: During the second phase of V(D)J recombination, RAG1 and RAG2 work together with DNA repair proteins to process and ligate coding ends to form a coding joint, and ligate signal ends to form a signal joint.  This phase requires ubiquitously expressed DNA repair factors of the non-homologous end joining (NHEJ) pathway. Nature Reviews Immunology 8, 302-312 (April 2008)

Products of V(D)J recombination: Roth, David B. "V (D) J Recombination: Mechanism, Errors, and Fidelity."Microbiology Spectrum 2.6 (2014).

V(D)J Recombination Errors: The two types of V(D)J recombination errors: Errors in Target Recognition Errors in End Joining

Errors in Target Recognition: In lymphoid neoplasms cryptic RSS(CRSS)：cRSSs capable of supporting recombination are present approximately once per kilo- base in random DNA sequences small size of RSS sequences; strict adherence of this sequence to consensus heptamer or nonamer sequence mismatching of RSSs and cRSSs that are adjacent to proto-oncogenes Roth D. 2014. V(D)J Recombination: Mechanism, Errors, and Fidelity

Errors in Joining: A pair of breaks created during normal V(D)J recombination are mistakenly joined to another break created by another mechanism Errors in joining involve events that join a RAG-mediated double-strand break to a broken DNA end created by a non-RAG-mediated mechanism Roth D. 2014. V(D)J Recombination: Mechanism, Errors, and Fidelity

Summary 1. V(D)J Recombination provide a method to create an almost limitless supply of different antibodies to target antigens associated with pathogens 2.Aberrent V(D)J Recombination like errors in target recognition and errors in End Joining, can play important roles in initiating oncogenic transformation

Reference https://www.youtube.com/watch?v=QTOBSFJWogE Roth D. 2014. V(D)J Recombination: Mechanism, Errors, and Fidelity Nature Reviews Immunology 8, 302-312 (April 2008) Schatz, David G., and Patrick C. Swanson. "V (D) J recombination: mechanisms of initiation." Annual review of genetics 45 (2011): 167-202.

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