Lecture 6 Flashcards
What is somatic diversification? What are the 3 steps?
Somatic diversification = altering Ig/TCR gene segments in a NON-heritable way
- Gene rearrangement (VDJ recombination)
- Somatic hypermutation
- Class switch recombination
Which region of the receptor gene does NOT undergo recombination?
C - constant region!
Which Ig/TCR chains have VD and J genes?
Heavy chain
Beta
Delta
Which Ig/TCR chains have V and J genes only?
Light chain (kappa and gamma)
Alpha
Gamma
Which Ig/TCR chains undergo recombination first?
Heavy chain, beta, delta
Makes sense - why these chains present first in the preT/B cell receptors!
Where does most diversity in receptors come from?
Junctional deletions/additions
Limited number of V, D, J genes - unlimited options for adding/deleting random nucleotides to the junctions between those genes
Does the V, D, or J gene code for CDR2 and 1? How does this affect the Ig/TCR function?
CDR1 and 2 - V gene
Directly coded for by this gene
Limited number of V genes in the DNA - limited diversity
Therefore, CDR1 and 2 interact with the MHC - less variable region
What is CDR3 coded by? How does this affect the Ig/TCR function?
V-J (D if there) junction
Junction = most diversity possible
Interacts with antigen bound by MHC
What molecules make up VDJ recombinase enzyme?
RAG 1 & RAG 2
What flanks either side of V/D/J genes?
RSS = recombination signal sequence
On either side of any gene that can undergo recombination
Heptamer - 12/23 bp - nonamer
What part of the RSS is highly conserved?
Heptamer - identical
Some nonamer variability
Lots of variability in the spacer
What is the difference betwe 12/23 spacer?
12 = 1 alpha helix turn in the CDR 23 = 2 turns
What is the point of having RSS?
Prevents recombination within the same locus
What is the 12/23 rule?
Rearranging gene segments are always flanked by different RSS lengths
12 matches with 23
Of the RAGs, which:
- Binds the RSS directly?
- Is catalytic?
- Facilitates binding via changes in affinity?
RAG 1 = binds RSS, cuts
RAG 2 = facilitates RAG1
Why is VDJ recombination cell specific?
B/c RAG1/2 only in IMMATURE lymphocytes
What are the 5 steps of VDJ recombination?
- RAG1/2 binds RSS - 2 RAG complexes total, 1 for each RSS
- RSS synapsis
- DNA cleavage
- Signal end joining
- Coding end joining
Explain RAG’s “two step” cleavage.
Cleavage in trans
1. Nick top strand
2. Hairpin formation
RAG stays bound to cut RSS
What are the 3 steps of coding joint formation? Think ENZYMES.
- Bind broken DNA ends via DNA-dependent protein kinase + ku70 + ku80
- End processing (additions or deletions to joint)
- DNA end ligation via DNA ligase 4
What is the DNA signal joint product that is cut out?
TREC = extrachromosomal circle
Which 2 enzymes can make additions to the joint?
Artemis - via hairpin or asymmetric opening - +s P nucleotides = DNA repair filling in short ends of uneven break with sequence that is palindromic to the original coding end Tdt - Non-templated addition
What aspect of NHEJ is unique to immature lymphocytes?
Tdt
What enzymes can make deletions to the joint?
DNA POL mu or gamma
What is the accessibility hypothesis?
Recombinase alone is not enough to initiate gene rearrangement
Needs access to the target genes
RAG2 has affinity for Ig/TCR loci - binds histones to open specific aspects of the DNA for RAG1
What are the general differences between RAG and NHEJ mutations/defects?
RAG mutation = defect in initiation of recombination
NHEJ mutation = defect in completion
What is Omenn syndrome? What do you see clinically in these patients?
RAG mutation - autosomal inheritance SCID + hyper-allergic reaction symptoms - RAG1/2 is diminished but not 100% gone - Biased few cells get made --> Th2 bias --> inflammation --> preferred B cell switch to IgE
How do you get SCID with radiation sensitivity?
Artemis/DNA ligase 4 mutations
No NHEJ in any cells
Do Ig or TCR genes undergo hypermutation and class switch? When do these occur?
Ig ONLY
Mature B cells - once bind antigen
What is somatic hypermutation? What is the goal of this process?
Point mutations in heavy or light chain V genes
Changes to the variable region
GOAL = Ab affinity maturation
What is class switch? What is the goal of this process?
Recombination of heavy chain C genes
Changes to constant region
GOAL = different Ab isotypes
Which enzyme initiates both SHM & CSR?
AICDA - induces DNA damage DNA repair: - BER - NMR = nucleotide mismatch repair - NHEJ
How do you get primary adult immunodeficiency?
Defected SHM & CSR
- AICDA mutation
- BER mutation
- NHEJ or DNA damage response gene mutations
What is AICDA’s mechanism?
Cytidine –> uridine
Creates C –> T transition
After AICDA, which is more likely to fix the damage vs propagate the error?
Fix = BER
Keep error intentionally –> point mutation to improve Ab binding site = NMR
What Ab isotype CANNOT undergo CSR?
IgD
What disease results from AICDA deficiency?
Type 2 hyper IgM syndrome
- autosomal
- mutations that inactivate AID
- recurrent bacterial infections, survive them but keep getting them b/c only making primary Ab
- No switching isotypes or high affinity Abs
What is type 1 hyper IgM syndrome?
CD40 or CD40L gene mutation
B cells can’t interact with T cells to induce AID expression
Symptoms:
- Repeated bacterial infections
- No germinal centers
- Immune deficiency b/c also lacking CD40 on macrophages
What disease could be attributed to VDJ recomb and CSR?
Cancer mutations - LEUKEMIA & LYMPHOMA
Due to chromosome translocations
