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what are the 3 mechanisms of somatic diversification in humans?

gene rearrangement, somatic hypermutation, class switch recombination (occur in lymphocytes and non-inheritable manner)


the variable domains of the antigen receptors is encoded by not a single, but multiple, discontinuous gene segments. The three kinds of segments are what?

variable (V), diversity (D), and joining (J) - together form the variable domain of Igs and TCR genes


What does the V gene encode?

the first two complementary determining regions (CDR1 and CDR2) and the first 3 framework regions (FR1, 2, and 3)


the D gene is only found in which genes?

IgH, TCRBeta, and TCRgamma


which CDR does the D gene contribute to?



what does the J gene encode?



what does the C gene encode?

constant domain (has multiple exons that are spliced together through txn but does not undergo rearrangement, the first exon of the C gene is also spliced but not rearranged to the J gene during txn)


key features of VDJ recomination?

" - site specific recombination (only operates at antigen receptor loci);
- lymphoid specific (only occurs in B and T-lymphocytes, at immature stage of differentiation);
- assembles only one V, one or more D (if available) and one J genes at the DNA level;
- absolutely required for the generation of functional antigen receptors and, therefore, for lymphocyte development.


what is IgH?

the heavy chain


CDR1 and CDR2 are encoded by which gene?

V gene (combinatorial diversity) (they match the MHC molecs)


CDR3 is encoded by which gene?

V(D) J junctions (junctional diversity)


VDJ recombination is initiated by which enzyme?

VDJ recombinase


VDJ recombinase is encoded by what genes?

RAG-1 and RAG-2 (the recombination activating genes)


What is the 12/23 rule?

rearranging gene segments are always flanked by different length RSS (recombination signal sequence) - V gene has 23 bp and is thus a 2 turn RSS between heptamer and nonamer, while J gene has 12 bp and is thus a 1 turn RSS between heptamer and nonamer. THE HEPTAMER AND NONAMER ARE THUS ALWAYS CONTACTED ON THE SAME SIDE OF THE DNA ***this is the reason why even one-two nucleotide differences in the length of the spacer abolish binidng and cause deficiency of B and T cells


RAG-1 vs RAG-2?

RAG-1 bind the RSS directly, catalytic DDE motif, ubiquitin ligase activity; RAG-2 associates with RAG-1, increases DNA binding affinity to the RSS, facilitates binding of RAG to the chromosome; both are absolutely required for VDJ recombination and both are specific for early, immature lymphocytes!


what occurs in the 1st phase of gene rearrangement?

DNA binding to RSS, 12/23 RSS synapsis, DNA cleavage (nicking/hairpin formation) by RAG-1/-2 proteins


what occurs in the 2nd phase of gene rearrangement?

DNA broken end binding, DNA hairpin/end processing, DNA joining (coding joint/signal joint) by NHEJ proteins (non homologous end joining repair of DNA ds breaks. Ubiquitously expressed activity in ALL CELLS and NOT LYMPHOID SPECIFIC)


what residues are the most important for RAG-1/RAG-2 binding?

nonamer residues (heptamer and conding end nucleotides are alos contacted but more important for cleavage than binding)


upon binding to the RSS, the RAG proteins bring what together?

the two RSS (one 12mer and one 23mer RSS) together and form a synaptic complex


what are the 2 steps of DNA cleavage in recombination?

1. nicking the top strand (precisely between the heptamer and the last nucleotide of the conding end of the gene segment) 2. hairpin formation (free 3'OH end from the top strand attacs the bottom strand and forms a hairpin on the coding end)….thus two broken DNA ends are generated: open, 5' phosphorylated RSS ends (signal ends) and hairpin-terminated coding ends. The four ends (two at each RSS-gene segment cleavage) remain in a complex with the rag proteins for a short time (four-end complex)


joining of the recombinant ends can only occur when?

when the RAG proteins release the ends


_____ are usually precise fusion of the first nucleotides of the heptamers of the excised RSS

signal joints


what happens to signal joints?

removed from the chromosome and become extrachromosomal circles that persist in mature lymphocytes - only lost during cell division because they do not replicate, therefore VDJ recombination excision circle (REC) can be used to monitor cell division history (rarely VDJ recombination occurs with inversion and the signal joint is retained on the chromosome)


the broken, hairpin-terminated coding ends are joined rapidly by the _______ repair enzyme complex.

NHEJ/non homologous DNA end joining double strand DNA break repair enzymatic complex


what make up the DNA-dependent protein kinase in recombination?

Ku70, Ku80, catalytic subunit (catalytic subumit is mutated in a rare human scid mutation)


what does the DNA-PKcs (catalytic subunit) of the NHEJ repair enzyme complex do in recombination?

binds the broken coding ends and recruits and activates other NHEJ enzymes


what opens up the hairpins in coding joint formation?



after artemis binds the coding ends and nucleases/polymerases modify the ends (deletions and insertions), what joins them?

DNA ligase4 protein complex


templated insertions in coding joint formation are called P-nucleotides and they are generated by what?

asymmetric opening of the hairpin ends (one strand is longer than the other, so filled in with palendromic sequence "P" by DNA polymerase)


template-independent insertions in coding joint formation are mediated by what enzyme?

terminal deoxynucleotidyl-transferase (Tdt) = a lymphoid specific enzyme that can add a few N-nucleotides to the end of broken DNA molecules iwthout the need for another strand (template)….only function is to modify the rearranged gene segments