V(D)J Recombination
N-Addition
(On heavy chain) With TdT - adding random nucleotides
Non-Productive Rearrangement
- If the RSSs are pointing away rom each other (deletional, yet found on the loop) - Could also be caused by frameshift
Antibody Diversity
- Junctional Flexibility (exonuclease trimming) - P-addition - N-addition - Somatic Mutation - Gene Conversion
RNA Processing
- Polyadenylation - Removal of Introns - Expression of more than one isotype at a time - Expression of membrane bound and secreted IgD
Function of RSS
- bring DNA together (nonamer and heptamer base pair) - Match parts (one turn with two turn) - Place for protein binding (VDJ recombinase)
Method of Rearrangement
1. Rearrange your Heavy Chain - D and J get together - Add the J on the front - Start expressing VDJ- Cm- Cd 2. Express Heavy Chain and Surrogate Light - Looks like light chain (not a full Ab)- B cell is looking at success of making heavy chain - If heavy chain doesn't work with surrogate chain, tries again (can try two times) - If after 2nd time it doesn't work- it dies 3. Light Kappa- always tries first 4. Light Lambda (mom or dad's)
V(D)J Recombination Steps
1. Recognition of heptamer-nonamer RSS by RAG1/RAG2 enzyme complex 2. RAG1/2 performs a single stranded nick at 5' border of the heptameric RSSs bordering both the V and J segments 3. Formation of V and J region hairpins and blunt signal ends (the hydroxyl group attacks the phosphate group of the non-coding strand of the V segment to yield a covalently-sealed hairpin coding end and a blunt signal end) 4. Ligation of the signal ends-- DNA ligase then ligates the free blunt ends to form the signal joint 5. Opening of the hairpin by Artemis-- can result in asymmetrical cleavage 6. Cleavage of the hairpin generates sites for P nucleotide addition 7. Ligation of light chain V and J regions (IN HEAVY CHAIN) 8. Exonuclease Trimming (occurs more in heavy chains than light-chain joints) 9. N-nucleotide addition (added by TdT to the coding joints of heavy-chain genes after hairpin cleavage) 10. Ligation and repair of the heavy-chain gene
P-addition
Artemis can cut anywhere (ss-endonuclease) - Cleaving the hairpin of coding strands- when done asymmetrically, palindrome addition
RAG1/ RAG2
First ones to sit down on RSS, holds the RSS in position (to prevent DNA from snapping back)- also has Exonuclease activity (used in junctional flexibility)
TdT
In heavy-chain genes, after hairpin cleavage- may add N nucleotides
Deletional Joining
Most common - If the RSS are facing each other 1. Combine those RSS pointing inward (one is double turn, one is single turn) 2. Once combined, it will remove RSS completely -- Cut off all RSS 3. Ligates RSS part to RSS part- results in loop that looks like a plasmid
Junctional Flexibility (exonuclease trimming)
RAG has exonuclease activity- has to start on an end (clip from artemis provides the end- can start chewing this up and can shorten the end) - joining anywhere (many parts can line up) - If frameshift = nonproductive
Components of RSSs
Recombinase Signaling Sequence - Heptamer - Nonamer - Spacers (single turn, double turn)
Enzymes Responsible for Rearrangement
VDJ Recombinase: - RAG1/ RAG2 - ss Endonuclease (Artemis) - Endonuclease - DSBR - Terminal Deoxynucletidyl Transferase (TdT) - Accessory Proteins
Inversional Joining
When RSSs are facing the same way (still one turn and two turn) 1. Begins by forming an s out of the DNA (taking the J and putting it right next to the V) 2. The RSSs are now close to each other and can connect- swing these around to form a loop (as seen with all RSSs) 3. Binding of boxes (the V and the J) so that they ligate, joining of RSSs - Complete rearrangement- we haven't lost any DNA (unlike deletional joining)
DSBR
double stranded break repair-- the ligase
Endonuclease
for blunt cut
ss Endonuclease (Artemis)
will clip one side and leave the other strand connected (V and J region hairpins -- there is a covalent bond across the double helix which must be cleaved by Artemis)- may be opened asymmetrically (leading to palindromic addition)
