Chapter 7 - immunology
3 sequence event in VDJ recombination
1- DH gene combine to JH 2- DHJH combine VH generate V domain 3- functional VL combines with funtional JLCL combinations
VDJ recombination series
1- RAG bind RSS, cleave DNA, unwind 2- other protein process hairpin loop that form after RAG react 3- Products of a recombined coding joint and a leftover signal joint that is later degraded
Nonamer conserved of
9- bp with sequense of 5'-ACAAACC-3'
combining of L and H chain can add
Final diversity of variable region
in VDJ recombination, J stand for ?
Joining segment
Role of Artemis generating diversity by adding P nucleotides
Once Artemis has been phosphorylated by DNA-PKcs, it opens the hairpin on the coding end joint.
Hairpin resolution results in
P-region nucleotide addition
Role of DNA-PKcs generating diversity by adding P nucleotides
Protein kinase that forms a complex with Ku70/80. It phosphorylates and activates Artemis, and recruits the ligation machinery VDJ recombination
what are 3 protein implicated in VDJ that are unique to lymphocytes?
RAG 1, Rag 2, and Terminal Dexynucleotidyl transferase (TdT)
What associated in gene segments joining?
RAG 1/2 recombinase
2 types of Rag
RAG1 & RAG2
High mobility group B proteins 1 and 2 (HMGB1/2)
Stabilize binding of RAG1/2 to recombination signal sequences (RSSs). Stabilize introduction of bend into 23 RSS DNA by RAG1/2
Where does TdT express and how it will express
TdT only happen at Lymphocytes and common in H chain. that can add up to 15-20 max nucleotides at cut end that create diversity
What is the segment for light chain?
V and J
What is the segment for heavy chain?
VDJ
List the 5 mechanisms that generate antibody diversity in naïve B cells
VDJ recombination, exonuclease trimming, P nucleotide addition, Non-templated nucleotide addition, combinatorial diversity
DNA ligation complex: DNA ligase IV, XRCC4, and XLF (Cernunnos)
XRCC4 maintains stability of DNA ligase IV and stimulates its catalytic activity. XRCC4 may also help to align DNA ends. DNA ligase IV is required for ligation of cut DNA ends, at both the coding and the signal joints.
how hairpin was introduce?
break near the tip of each hairpin in one of the DNA strands. Cutting of the hairpins generates sites for the addition of P-region nucleotides which adds to diversity.
Exonuclease trimming
common in 2 H chain V gene joints (V-D & D-J) than at L chain V-J joint. result in loss of coding nucleotide at joint
3 element in RSSs
conserved of "nonamer & heptamer", spacer 12 or 23 bp sequence, 12 bp RSSmust pair with 23bp RSS for recombination to occur (12/23rule)
TdT functions
develop only in lymphocytes. Adds nontemplated (N) nucleotides to V-D and D-J joints of Ig heavy chain and all joints of TCR chains in a template independent manner
What are the VDJ recombination?
directed by Recombination signal sequences (RSSs) that ensure each type of sequence is included in variable region gene
unique of RAG 1
express in all phases of cell cycle
hairpin formation
free 3'-OH on the cut DNA strand attacks the phosphodiester bond linking the opposite DNA strand to the SS, simultaneously producing a hairpin structure at the cut end of the CS & a flush, 5'-phosphorylated, double-strand break at the SS.
purpose of cutting hairpin
generates sites for addition of P-region nucleiotides
Why RAG1 are more more important
it form complex with RSS to stabilized biding with RAG2
Exonuclease trimming AKA
junctional flexibility
Non-templated N nucleotide addition are
mediated by TdT activity, adding in random nucleotides between joints
VDJ recombination are
of multiple gene segments
Function of hairpin structure
protects gene segment from being degraded, prevent them from being joined together immediately into functional gene
What doe RAG1/2 responsible for
recognizing and cutting DNA at immunoglobulin-encoding and the RSS
What is RAG?
recombination activating gene
both Rag 1/2 developed by
regulated in both T and B cells
what are 3 protein function in VDJ?
responsible for the generation of additional diversity in CDR 3 (complementary determining region) of the antibody heavy chain
combinatorial diversity are
same H chain can combine with different L chains, and vice-versa
unique of RAG 2
stable only in G0 or G1 phase cell
P nucleotide addition are
templated nucleotide addition between joints, resulting from assymetrical cleaving of hairpin structures
Role of TdT to generate diversity
the addition of N-nucleotides.
Exonuclease trimming are
trimming―sometimes occurs at joints, causing loss of nucleotides and changed reading frames
in VDJ recombination, V stand for?
variable region
define combinatorial diversiry
which heavy chains pair with which light chains
In human, what are the possible number of potential VDJ combination in Lambda light?
33 x 5= 165 possible VDJ combinations
In human, what are the possible number of potential VDJ combination in Kappa light?
41 x 5= 205 possible VDJ combinations
In human, what are the possible number of potential VDJ combination in heavy chain?
45 x 23 x 6= 6210 possible VDJ combinations
Heptamer conserved of
7- bp with sequence of 5'-CACAGTG-3'
who can form complex hairpins
DNA-dependent protein kinase (DNA-PK) and Artemis protein
in VDJ recombination, D stand for ?
Diversity segment