Site Specific Recombination and Transposition

Question 1

What are the two ways by which discrete segments of DNA can move from one region of the genome to another?

Answer 1

1) Site-specific recombination 2) Transposition

Question 2

Site Specific recombination structure.

Answer 2

a) CSSR conservative site-specific recombination (CATACA) recombination site. (b) Flanked by Cre binding sites that aid in recombination protein binding

Question 3

Recombinase proteins

Answer 3

bind to the cre binding site. cleave phosphodiester backbone of DNA. Act like topoisomerases (no ATP involved)

Question 4

What is the only way that SSR will occur?

Answer 4

the region of strand exchange between the two DNA molecules must be in the same direction.

Question 5

Direction oreintation

Answer 5

sites are in the same direction - leads to excision

Question 6

sites in inverted oreintation

Answer 6

sites are not in the same direction - leads to inversion

Question 7

Mechanism of site specific recombination

Answer 7

1. regions of strand exchange line up 2. recombinase does nucleophilic attack on phosphodiester backbone (tyrosine leads to 5’OH formation, while serine leads to 3’OH formation) 3) tyro-phosphate groups and OH groups on opposite strands attract. 4) other set of topoisomerases produce OH and tryophosphates which combine and repair to form two different strands.

Question 8

SSR on the same DNA molecule

Answer 8

If rec sites are in opposed direction, inversion; if rec sites are in the same direction you get an insertion and deletion.

Question 9

Transposition

Answer 9

jumping genes, short sequences (transposons) can excise and reinsert at a different place in the genome.

Question 10

Inverted repeats

Answer 10

reads one way L-R and the same way R-L on a different site on DNA

Question 11

Transpoase gene

Answer 11

encodes transpoases; transposases recognize IRs, cut piece of DNA out of site, and move it to different site.

Question 12

mobile genetic element

Answer 12

can move from one region to another

Question 13

Flanking DNA

Answer 13

regions on target DNA that recognize ends of transposons ( terminal repeats). gaps mad when transposon and target DNA are fixed by DNA repair mechanism. The flanking DNA act as templates.

Question 14

Direct transposition

Answer 14

(DS breaks) 1) Cleavage of transposons 2) Free ends of transposons attack target DNA 3) Gaps filled (left) by DNA polymerase and ligase

Question 15

Replicative transposition

Answer 15

1) Cleavage of transposons 2) Free ends of transposons attack target DNA 3) entire transposon replicated 4) Site specific recombination (with in the transposon) DNAP III mediated replication,

Question 16

What type of recombination produces genetic diversity in adaptive immunity?

Answer 16

Site specific recombination during assembly of Ig genes.

Question 17

VJ recombination

Answer 17

Occurs in light chain

Question 18

VDJ recombination

Answer 18

process

Question 19

VJ recombination process

Answer 19

1) RSS sequence associated with V segment and J segment 2) Recombinase binds to RSS sequence on V and J. 3) Rag1/2 form 3’OH and the 3’OH does a nucleophilic attack on its own strand watson-crick strand. 4) The genetic information in the middle gets removed.

Question 20

VDJ Recombination

Answer 20

1) RAG 1/2 binds to V and D RSS 2) cleavage produces 3’OH 3) 3’OH attacks own watson crick strand 4) Ku70/80 involved in joining. Artemis opens hairpins and activates Ku70/80,