DNA Recombination

Question 1

The steps of Double Strand Break Repair

Answer 1

1. 5’ ends of each strand are chewed back.
2. 3’ overhangs from each end invade undamaged strand which results in a displacement loop (D-loop).
3. Elongation (DNA Polymerase adds nucleotides).

Question 2

The Two Resolutions of DSBR

Answer 2

1. SDSA (Synthesis Dependent Strand Annealing)- helicases free intertwined strands
2. Holliday Junction Resolution

Question 3

Holiday Junction Resolution

Answer 3

A Class of endonucleases called Holliday junction resolvases facilitate the resolution

X/X & Y/Y resolutions (same kind of cleavage) result in the non-cross over product

X/Y & Y/X (cleavage in different directions) result in the cross-over product

Question 4

What is the significance of the D-loop that forms after strand invasion?

Answer 4

D-loop provides the template for the Double Strand Break Repair

Question 5

Draw a DSBR Mechanism

Question 6

Draw the final products after the resolution of holiday junction through x/x and x/y cleavage.

Question 7

What form of recombination is DSBR?

Answer 7

Homologous Recombination

Question 8

What are the two important roles that homologous recombination plays in meiosis?

Answer 8

1. creation of genetic diversity via the interchange of DNA.
2. Segregation in Meiosis I: Links together homologous chromosomes to accomplish the goal of segregating one chromosome of each type into a daughter cell.

Question 9

What is VDJ Recombination?

Answer 9

it is a way to create antibody diversity.

Question 10

What are the steps of VDJ Recombination?

Answer 10

1. Binding of RAG1/2, HMG to bind the signal sequences.
2. Synapsis: Two sequences are made adjacent to each other via the 12/23 rule.
3. All of the signal sequences are cleaved off, remaining DNA ends are joined into a single stranded hairpin.
4. circular intermediates are then cut to restore dsDNA.

Question 11

What are signal sequences?

Answer 11

short stretches of DNA that flank the gene segments used in VDJ recombination.

Question 12

What is the 12/23 rule?

Answer 12

when two different gene segments are brought together by the rag enzymes, they must pair a 23 spacer sequence with a 12 spacer sequence.

Question 13

Why is the 12/23 rule important?

Answer 13

create a restriction on the pairing of gene segments during VDJ recombination that is essential for generating a diverse but functional antibody.

Question 14

What is importance of NHEJ?

Answer 14

primary DNA repair mechanism that mediates VDJ recombination by joining the broken ends of the DNA segments together.

Question 15

How does NHEJ contribute to the diversity of antibodies?

Answer 15

introduction of small deletions or insertions at the junctions.

Question 16

What is Tdt?

Answer 16

Tdt polymerase adds random nucleotides during the process, increasing the variability of recombination.

Question 17

What is a coding joint?

Answer 17

formed by the precise joining of the coding ends.

Question 18

What is the signal joint?

Answer 18

(unused DNA) formed by the imprecise joining of the signal ends. Not retained in final antigen receptor.