VDJ Recombination Flashcards
Germ-line Theories
Entire geneomic information to generate Abs in present in the germ-line genome (Ab diversity is generated bt recombining seperate DNA segments in the germ-line genome of B cells)
Somatic mutation theories
Ab variablility reigons undergo mutation in B cells to generate a diverse repertoire of Ab
Which of the 2 theories (germ-line and somatic mutation) are true?
Both!
What gene segments make up the light chain of an Ab?
V (variable) and J (joining) segments
What gene segments make up the heavy chain of an Ab?
V (varibale), D (diversity) and J (Joining) segments
Combinatoral diversity (what is the process that allows for this called?)
The combination of different V and J (LC) segements and V, D, and J (HC) segments to allow for fifferent LC and HC’s to be generated
the process of doing this is called V(D)J recombination
What is the benifit of having multiple V, D, and J segements?
Allows for many combinations to be created from the multiple copies of each segment
What 3 factors contribute to combinatoral diversity?
- VDJ recombination in the HC
- VJ recombination of the LC
- The combination of the HC and LC
RSS (recombination signal sequence)
- critical for recombination
- they are heptamer or nonamer sequences that are conserved with respect to size and sequence and are the sequence before each V, D and J segment
- there are spacers between each RSS that are conserved in respect to size but NOT sequence
12/23 rule
RSS sequences with 12bp spacers can only recombine with RSS sequences with 23bp spacers
aka… 1turn/2turn rule
Describe the recombination of the kappa LC?
- 3’ of each Vk (kappa) segement is a 12bp RSS and each Jk segment has a 5’ 23bp RSS
- 1 V segment recombines with 1 J segment to generate VL (varibale light) of the kappa light chain and intervening DNA is removed
Describe the recombination of the HC?
- 3’ of each V segment is a 23bp RSS, each J segment has a 5’ 23bp RSS, and the D segment has a 5’ and 3’ 12bp RSS motif
- 1 D segment first recombines with 1 J segment, then 1 V segment recombines with the recombined DJ segment to generate the VH reigon and the intervening DNA is removed in both cases
Recombinase activating genes 1/2 (RAG-1/2)
- lymphoid specific proteins that catalyze DNA strand breakage
- absolutley required for VDJ recombination (including TCR gene rearrangments)
high mobility group box (HMGB) proteins
Stabilize RAG-1/2 binding and help facilitate the bending of DNA
Describe how RAG-1/2 contributes to VDJ recombination.
the RAG-1/2 proteins nick the 5’ end of the heptameric RSS sequences. There s a free hydroxyl (-OH) groupcreated by the nick that will attack the phosphate on the opposite strand creating a hairpin structure.
The free hydroxyl group binds to the opposite strand with the phosphate forming the coding end (this is the combined V(D)J segment depending on what we are talking about.
the RSS sequences that did not bind are the signalling ends
(see lecture notes if confused)
How does DNA ligase IV contribute to VDJ recombination?
It joins the 2 signal ends created from the RAG-1/2 action. The ligation results in the joining of the 2 heptameric RSS reigons (a circular episome) which is lost as the B cell divides during development
Artemis
Endonuclease that nicks the hairpin at one spot
Describe how artemis contributes to VDJ recombination.
- It has different outcomes depending on the nick site…
Can make a blunt end, 5’ overhangs, or 3’ overhangs (most common) - Cleavage events that generate overhangs can be filled in which P (palindromic nucleotides)
- Artemis with the help of DNA-PKcs (protein kinases) remove nucleotides through its exonuclease activity
DNA pol mu
Adds non-templated (N) nucleotides between the joints
What mechanisms contribute to Junctional Diversity?
- P nucleotide addition through Artemis
- endonuclease activity of Artemis/DNA-PKcs
- N nucleotide addition by DNA Pol mu
- exonuclease trmming
- N nucleotideaddition by TdT
What mechanisms contribute to junctional diversity in the HC?
- Deletion of nucleotides by an exonuclease
- Addition of N (non-templated) nucleotides by TdT (terminal deoxynucleotidyl transferase). There can be up to 20 nucleotides added at each end
How does VDJ recombination effect the Ag binding sites of Abs?
- VDJ and VJ recombination alters the sequence of CDR3
- CDR1 and 2 are in the V segment
What factors of combinatoral/ junctional diversity effect the CDR1 and CDR2?
V segment divrsity and somatic hypermutation
What factors of combinatoral/ junctional diversity effect the CDR3?
V segment diversity, VDJ recom, Artemis/DNA-PKcs, Endonuclease activity, DNA pol mu templated nucleotide addition, exonuclease trimming, P-nucleotide addition, N-nucleotide addition, and somatic hypermutation
Compare the VDJ recombination process from BCRs to TCRs?
- they are the same overall process
- the alpha chain in the TCR is similar to the light chain and has V and J segments
- the beta chain in the TCR is similar to the heavy chain
- the BIGGEST difference is that receptor genes undergo somatic hypermuation following the antigenic stimulation
Explain the whole process of VDJ recombination.
- RAG-1/2 and HMGB-1/2 bind to the RSS and catalyze synapse formation between V and J gene segemnt
- RAG-1/2 performs a ss nick at the exact 5’ border of the heptameric RSSs bordering both the V and J segments
- The hydroxyl grroup that was liberated by the nick at the 3’ end of the coding strand attacks the corresponding phosphate group on the noncoding strands of both the V and J segments to yeild a covalently sealed hairpin coding end and a blunt signal end
- Signal end joining ligates the ends of the 2 RSS heptameric sequences that were originally in contact with the V and J coding sequences
- Opening of he hairpin can result in a 5’ overhang, a 3’ overhang (most common), or a blunt end
- Cleavage of the hairpin generates sites for P nuclotide addition
- Ligation of the LC V and J reigons
***Steps 8-10 only happen in HC - Exonuclease cleavage can result in the loss of nucleotides on either or both sides of the coding end
- nontemplated nucleotides are added to the coding joint by the TdT or occasionally by pol mu
- Ligation of the heavy chain my NHEJ DNA ligase complex
