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
What is the tenth step in V(D)J recombination (note this only happens in the heavy chain)?
ligation of heavy chain
How does V(D)J recombination effect Ag binding sites of Ab.s?
most diversity (combinatorial/junctional) occurs in changing CDR3, CDR1 and 2 are within the V segments
What processes result in changing CDR3?
- V segment diversity
- V(D)J recombination
- exonuclease trimming
- P-NT addition
- N-NT addition
- somatic hypermutation
What process result in changing CDR1 and 2?
- V segment diversity
- somatic hypermutations
Briefly describe V(D)J recombination in TCRs
- alpha (gamma) is similar to light chain and has V and J segments
- beta (delta) is similar to heavy chain and has V, D, and J segments
- TCR alpha and delta chains are on the same chromosome (delta completely within alpha region) which is important in development -> lineage differences -> differentiation
Describe how TCRs follow the 12/23 rule
- V region segments contain a 3’ 23bp RSS
- D region segments contain a 5’ 12bp RSS and a 3’ 23bp RSS
- J region segments contain a 5’ 12bp RSS
Where does V(D)J recombination generate the most diversity in a TCR?
CDR3 in both the alpha and beta chain, CDR1 and 2 are within the V segments
Where does junctional diversity arise from in V(D)J recombination?
- P- NT addition
- exonuclease trimming
- N-NT addition
