Genome Instability II

Question 1

4 steps of VDJ recombination

Answer 1

binding, synapsis, cleavage, and joining - be able to draw

Question 2

proteins involved in VDJ recombination

Answer 2

RAG1/2, NHEJ proteins (Ku 70/80, Artemis, DNA-PKcs, ligIV, XRCC)

Question 3

preferred pathway for repair in G1

Answer 3

NHEJ, no sister chromatid for HR

Question 4

2 proteins needed for coding joint but not signal joint formation, and why?

Answer 4

Ku, DNA-PKcs. Bc signal joint already ds breaks, coding joint is hairpins

Question 5

how is RAG recombination separated from HDR?

Answer 5

By cell cycle - RAG2 only expressed in G1, whereas RAG1 has stable levels throughout cell cycle

Question 6

4 similarities between RAG1 catalytic site and transposase?

Answer 6

1. DDE motif
2. Zn as cofactor
3. binds/cleaves in trans
4. RNase H fold bringing catalytic residues to proximity

Question 7

Why doesn’t RAG attach V-V to D-D?

Answer 7

RAG1 can only work if DNA segments are of different lengths - can only accommodate a longer seq on left bc of flexible linker that can rock back and forth

Question 8

draw an antibody

Answer 8

parts to know/label: heavy chain, light chain, antigen binding site, constant region, variable region

Question 9

type I, II, III CRISPR differences

Answer 9

Type I: Cascade recognizes PAM, recruits Cas3 to cut
Type II: Cas9, tracrRNA, pre-crRNA (RNase III activity)
Type III: Cas6 processing, cas10 displaces, cleavage only happens as transcription occurs

Question 10

why doesn’t Cas9 cleave itself?

Answer 10

PAM

Question 11

what are 4 ways bacteria have developed resistance to phage

Answer 11

restriction digest enzymes, prevention of phage adsoption/injuction, cell suicide and CRISPR

Question 12

what are 2 phases of CRISPR activity?

Answer 12

spacer acquisition, immunity/interference phase

Question 13

spacer acquisition mechanism

Answer 13

be able to draw, involves two nucleophilic attacks andn integration