Lecture 6

Question 1

What is somatic diversification? What are the 3 steps?

Answer 1

Somatic diversification = altering Ig/TCR gene segments in a NON-heritable way

1. Gene rearrangement (VDJ recombination)
2. Somatic hypermutation
3. Class switch recombination

Question 2

Which region of the receptor gene does NOT undergo recombination?

Answer 2

C - constant region!

Question 3

Which Ig/TCR chains have VD and J genes?

Answer 3

Heavy chain
Beta
Delta

Question 4

Which Ig/TCR chains have V and J genes only?

Answer 4

Light chain (kappa and gamma)
Alpha
Gamma

Question 5

Which Ig/TCR chains undergo recombination first?

Answer 5

Heavy chain, beta, delta

Makes sense - why these chains present first in the preT/B cell receptors!

Question 6

Where does most diversity in receptors come from?

Answer 6

Junctional deletions/additions
Limited number of V, D, J genes - unlimited options for adding/deleting random nucleotides to the junctions between those genes

Question 7

Does the V, D, or J gene code for CDR2 and 1? How does this affect the Ig/TCR function?

Answer 7

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

Question 8

What is CDR3 coded by? How does this affect the Ig/TCR function?

Answer 8

V-J (D if there) junction
Junction = most diversity possible
Interacts with antigen bound by MHC

Question 9

What molecules make up VDJ recombinase enzyme?

Answer 9

RAG 1 & RAG 2

Question 10

What flanks either side of V/D/J genes?

Answer 10

RSS = recombination signal sequence
On either side of any gene that can undergo recombination
Heptamer - 12/23 bp - nonamer

Question 11

What part of the RSS is highly conserved?

Answer 11

Heptamer - identical
Some nonamer variability
Lots of variability in the spacer

Question 12

What is the difference betwe 12/23 spacer?

Answer 12

12 = 1 alpha helix turn in the CDR
23 = 2 turns

Question 13

What is the point of having RSS?

Answer 13

Prevents recombination within the same locus

Question 14

What is the 12/23 rule?

Answer 14

Rearranging gene segments are always flanked by different RSS lengths

12 matches with 23

Question 15

Of the RAGs, which:

• Binds the RSS directly?
• Is catalytic?
• Facilitates binding via changes in affinity?

Answer 15

RAG 1 = binds RSS, cuts

RAG 2 = facilitates RAG1

Question 16

Why is VDJ recombination cell specific?

Answer 16

B/c RAG1/2 only in IMMATURE lymphocytes

Question 17

What are the 5 steps of VDJ recombination?

Answer 17

1. RAG1/2 binds RSS - 2 RAG complexes total, 1 for each RSS
2. RSS synapsis
3. DNA cleavage
4. Signal end joining
5. Coding end joining

Question 18

Explain RAG’s “two step” cleavage.

Answer 18

Cleavage in trans
1. Nick top strand
2. Hairpin formation
RAG stays bound to cut RSS

Question 19

What are the 3 steps of coding joint formation? Think ENZYMES.

Answer 19

1. Bind broken DNA ends via DNA-dependent protein kinase + ku70 + ku80
2. End processing (additions or deletions to joint)
3. DNA end ligation via DNA ligase 4

Question 20

What is the DNA signal joint product that is cut out?

Answer 20

TREC = extrachromosomal circle

Question 21

Which 2 enzymes can make additions to the joint?

Answer 21

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

Question 22

What aspect of NHEJ is unique to immature lymphocytes?

Answer 22

Tdt

Question 23

What enzymes can make deletions to the joint?

Answer 23

DNA POL mu or gamma

Question 24

What is the accessibility hypothesis?

Answer 24

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

Question 25

What are the general differences between RAG and NHEJ mutations/defects?

Answer 25

RAG mutation = defect in initiation of recombination | NHEJ mutation = defect in completion

Question 26

What is Omenn syndrome? What do you see clinically in these patients?

Answer 26

``` 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 ```

Question 27

How do you get SCID with radiation sensitivity?

Answer 27

Artemis/DNA ligase 4 mutations | No NHEJ in any cells

Question 28

Do Ig or TCR genes undergo hypermutation and class switch? When do these occur?

Answer 28

Ig ONLY | Mature B cells - once bind antigen

Question 29

What is somatic hypermutation? What is the goal of this process?

Answer 29

Point mutations in heavy or light chain V genes Changes to the variable region GOAL = Ab affinity maturation

Question 30

What is class switch? What is the goal of this process?

Answer 30

Recombination of heavy chain C genes Changes to constant region GOAL = different Ab isotypes

Question 31

Which enzyme initiates both SHM & CSR?

Answer 31

``` AICDA - induces DNA damage DNA repair: - BER - NMR = nucleotide mismatch repair - NHEJ ```

Question 32

How do you get primary adult immunodeficiency?

Answer 32

Defected SHM & CSR - AICDA mutation - BER mutation - NHEJ or DNA damage response gene mutations

Question 33

What is AICDA's mechanism?

Answer 33

Cytidine --> uridine | Creates C --> T transition

Question 34

After AICDA, which is more likely to fix the damage vs propagate the error?

Answer 34

Fix = BER | Keep error intentionally --> point mutation to improve Ab binding site = NMR

Question 35

What Ab isotype CANNOT undergo CSR?

Answer 35

IgD

Question 36

What disease results from AICDA deficiency?

Answer 36

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

Question 37

What is type 1 hyper IgM syndrome?

Answer 37

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

Question 38

What disease could be attributed to VDJ recomb and CSR?

Answer 38

Cancer mutations - LEUKEMIA & LYMPHOMA | Due to chromosome translocations