Adaptive Immunity in Vertebrates

Question 1

How does RAG-1 and RAG-2 generate double strand breaks?

Answer 1

The coding strand is nicked by enzyme.

3’ OH can attack non-coding strand and lead to hairpin termination end.

Question 2

Why are D600 and D708 essential for RAG catalysis of DNA cleavage?

Answer 2

These two amino acids facilitate coordination of divalent cations (Zn) in the catalytic active site.

Question 3

What is the relationship between the binding site and cleaving site in RAG-1?

Answer 3

In trans. binding and cleaving site are done by separate domains.

Question 4

What kind of fold does RAG-1 assume in its active site?

Answer 4

RNase H fold

Question 5

How does RAG-1 know know to combine V-J domains instead of combining V-V domains?

Answer 5

Region that binds DNA can rock back and forth due to being connected to the rest of the protein by a linker. If a long piece of DNA binds one end, then the DNA binding regions kinks and can only bind a short piece of DNA.

Question 6

How does V(D)J recombination seal ends once RAG1 and RAG2 facilitate hairpin termination end formation?

Answer 6

Uses enzymes involved in NHEJ.

Question 7

Function: Ku70/Ku80

Answer 7

Heterodimer that binds DNA ends in NHEJ and V(D)J recombination

Question 8

Function: XRCC4

Answer 8

Stimulates DNA Ligase IV

Question 9

Function: DNA-PKcs

Answer 9

Associates with Ku70/Ku80 and gets stimulated by it. Important for formation of coding joints.

Question 10

In VDJ recombination, what is the coding joint and signal joint?

Answer 10

Signal joint is the region between two gene segments (region between V and D, D and J) that gets cut out during rearrangement. The coding joint is the combination of VD and DJ.

Question 11

What sequence in the DNA is recognized by RAG-1 and RAG-2?

Answer 11

the RSS (recombination signal sequence)

Question 12

What are the 5 similarities between RAG-1 and transposases?

Answer 12

1. Have conserved DDE motif in each subunit
2. Utilize divalent cation for mechanism
3. DNA binding and cleavage are done in trans by the protein
4. Both proteins have an RNAse H fold
5. Nicking causes exposed 3’ OH that can perform nucleophilic attack.

Question 13

Function: Artemis

Answer 13

DNA-PK-dependent nuclease. Phosphorylated by DNA-PK and cleaves hairpin then trims ends.

Question 14

Function: XRCC4

Answer 14

Activates ligase IV

Question 15

Function: Ligase IV

Answer 15

Ligates the two blunt ends in NHEJ

Question 16

Knocking out which proteins causes signal joint defects?

Answer 16

Ku70, Ku80, Ligase IV, XRCC4

Question 17

Knocking out which proteins causes coding joint defects?

Answer 17

All of the NHEJ proteins?

Question 18

Why does knocking out Artemis or DNA PK affect coding joint formation but not signal joint formation?

Answer 18

In coding joint formation, the hairpin on each end of the DNA has to be cleaved. In signal joint formation, RAG is already bound to double strand breaks so it does not require Artemis endonuclease activity.

Question 19

How is the expression of RAG regulated in the cell cycle?

Answer 19

RAG-2 is upregulated in G1 when NHEJ is common. RAG-2 is subsequently degraded in the G1-S Transition

Question 20

Describe the mechanism that couples V(D)J recombination to the cell cycle through RAG-2 degradation.

Answer 20

1) During the G1-S transition, CDK2/Cyclin A is active.
2) CDK2/Cyclin-A phosphorylates RAG-2 at T490
3) Multiple proteins associates with phosphorlyated RAG-2
4) Cdc34 polyubiquitinates RAG-2 and targets RAG-2 for proteosome cleavage

Question 21

What happens if you prevent RAG-2 from being degraded?

Answer 21

There will be multiple chromosomal translocations. You also need to mutate p53.

Question 22

What are 4 ways that bacteria confer resistance against bacteriophages?

Answer 22

Prevent phase adsorption/injection of genome
Abortive infection by cell death
Restriction-modification
CRISPR

Question 23

What is the mechanism of bacterial spacer integration?

Answer 23

1) Cas1-Cas2 targeting complex bind DNA fragments from viral genome with PAM motifs.
2) DNA fragment is chewed up to lose all but one nucleotide of PAM and expose 3’ ends
3) 3’ OH does sequential nucleophilic attacks on junction between spacer and repeat sequence.
4) Filling in the gaps causes repeats to flank protospacer sequence.