BIOL 435 Ch. 6 V(D)J Recombination Steps

Question 1

V(D)J recombination steps

Answer 1

1. Recognition of the recombination signal sequence (RSS) by RAG1/2
2. One-strand cleavage at jucntion of the coding and signal sequences
3. Formation of V and J region hairpins and blunt signal ends
   4 .Ligation of the signal ends
4. Hairpin cleavage
5. Overhang extension can lead to addition of palindromic (P) nucleotides
6. Ligation of light chain V and J segments
7. Exonuclease trimming
8. N-nucleotide addition
9. Ligation and repair of heavy chain gene

Question 2

recognition of RSS by RAG1/2

Answer 2

-forms a complex with RSS next to one of two gene segments to be joined
-binding enhanced by HMGB1/2 proteins
>also facilitates bending of DNA
-2nd RSS bound by RAG1/2 and both segments are brought close together

Question 3

one strand cleavage at juction

Answer 3

-RAG1 creates ss nicks
>5’ of the heptameric signal sequence on the coding strand of each V-segment (b/w the V-segment and heptamer)
>clevage at heptamer J-region junction

Question 4

formation of V and J region hairpins and blunt signal ends

Answer 4

-free 3’-OH group at end of coding strand of V-segment attacks phosphate group on opposite non coding V-strand
>forms a covalent phosodiester bond across the double helix, yeilding a DNA hairpin =coding end

Question 5

ligation of the signal ends

Answer 5

-NHEJ protein, DNA ligase IV, ligates teh free blunt end to form the signal joint
>results from joining of the 2 heptameric regions

Question 6

hairpin clevage

Answer 6

-hairpins at ends of V and J are opened by Artemis in 1 of 3 ways

Question 7

3 cuts of Artemis

Answer 7

1. Blunt end at coding joint
2. Top: 5’ overhang
3. Bottom: 3’ overhang
   >most common
   >leaves 2 unpaired residues

Question 8

overhang extension

Answer 8

• can lead to addition of palindromic (P) nucleotides

- can occur at V-D and D-J and V-J

Question 9

ligation of light chain V and J segments

Answer 9

• by DNA ligase IV

* NHEJ repair mechanisms can generate nucleotide diversity

Question 10

exonuclease trimming

Answer 10

• trims back edges of V region
• more common at V-D, D-J in heavy chain
• extensive could lead to complete loss of D region and any P-nucleotides

Question 11

N-nucleotide addition

Answer 11

-nontemplated (N) nucleotides added by TdT to coding joints of H-chain genes after hairpin clevage

Question 12

ligation and repair of H-chain gene

Answer 12

• mediated by DNA ligase IV

- identical to ligation and repair in light-chain genes