V(D)J Recombination Flashcards
What are the two theories of how a genome of finite size can generate 10^14 Ab.s of different specificities?
- germ line theory
- entire genetic info to generate Abs is present in the germ-line genome
- Ab diversity generated by recombining separate DNA segments in the germline genome of a B cell
- applies to B and T cells - somatic mutation theory
- Ab variable regions undergo mutations in B cells to generate a diverse repertoire of Ab.s
- applies to only B cells
What is the Dreyer and Bennett hypothesis?
Regions coding for the V and C regions of Ab.s are brought together through recombination of DNA in B cells - somatic recombination
What did Hozumi and Tonegawa show experimentally?
That DNA encoding for Lv and Lc regions are far apart in non-B cells and brough together in developing B cells - evidence of somatic recombinations
What are a couple of characterisitics of light and heavy chain gene segments?
- segments are quite far apart
- each variable gene has a leader sequence in front of it
What is the purpose of a leader sequence?
ER localization signal
What segments do light chains have and which segments do heavy chains have?
light = V and J
heavy = V, D and J
Which segment is the major region of Ag-binding
V segment
What is V(D)J recombination and what kind of diversity does it create?
V(D)J = combining different V and J (light chain) or V, D and J (heavy chain) segments allow different heavy and light chains to be generated
diversity = combinatorial diversity
What two factors contribute to combinatorial diversity?
- V(D)J recombination
- combining of heavy and light chains
What is the recombinational signal sequence (RSS)
- at least one RSS flanks each V, D, and J segment
- critical for recombination
- conserved heptamer-spacer-nonamer sequence
How are heptamers, nonamers, and spacers conserved in the RSS?
heptamers and nonamers = conserved with respect to size and sequence
spacers = only size is conserved, either 12bp or 23bp
What is the 12/23 rule?
RSS sequences with 12bp spacers only recombine with RSS sequences with 23bp spacers (also known as one turn/two turn rule)
How is the kappa light chain recombined?
- 3’ of each Vkappa segment is a 12bp RSS, each Jkappa segment has a 5’ 23bp RSS
- one V segment recombines with one J segment to generate to V(L) of the kappa light chain
- intervening DNA is removed
How is the heavy chain recombined?
- 3’ of each V segment is as 23bp RSS, each J segment as a 5’ 23bp RSS, D segments have a 5’ and 3’ 12bp RSS
- one D segment recombines with one J segment
- one V segment recombines with the recombined DJ segment to generate the V(H) region
- intervening DNA is removed in both cases
What is recombinase activating genes - 1/2 (RAG-1/2)?
- lymphoid specific proteins that catalyze DNA strand breakage
- required for V(D)J recombination (including TCR gene rearrangement)
- high mobility group box (HNGB) proteins stabilize Rag-1/2 binding and help facilitate bending of DNA
What is the first step in V(D)J recombination?
Rag-1/2 and HNGB proteins bind to the RSS and catalyze synapse formation between a V and a J segment (bends the DNA)
What is the second step in V(D)J recombination?
Rag-1/2 proteins nick 5’ of the sequence of the heptameric RSS sequence bordering both the V and J segments
What is the third step in V(D)J recombination?
Free hydroxyl group created by nick attacks phosphate on opposite strand and creates a convalently sealed hairpin coding end and a blunt signal end. This happens at both the V and J segments.
What is the fourth step in V(D)J recombination?
Ligation of the heptamer sequences by DNA ligase IV to create a signal joint. The circular episome (intervening DNA) is lost as a B cell divides during development
What is the fifth step in V(D)J recombination?
Artemis (endonuclease) with the help of other proteins nicks hairpin at one spot, either creating a 5’ overhang, 3’ overhang (most common) or a blunt end. Where the strand is nicked affects the nucleotides that are added -> diff a.a -> variability
What is the sixth step in V(D)J recombination?
cleavage events that generate overhangs can be filled in with P (palindromic) nucleotides -> junctional diversity
What is the seventh step in V(D)J recombination?
Ligation of the light chain V and J regions
What is the eighth step in V(D)J recombination (note this only happens in the heavy chain)?
Deletion of nucleotides by an exonuclease - exonuclease trimming -> junctional diversity
What is the ninth step in V(D)J recombination (note this only happens in the heavy chain)?
addition of up to 20 N (non-templated) nucleotides by TdT (terminal deoxynucleotidyl transferase) which is not expressed during L chain reassortments -> junctional diversity
