Lecture 15 Reading

Question 1

Transposons

Answer 1

Mobile elements can move between cells through horizontal gene transfer.

The spread of antibiotic resistance is largely due to genes that encode antibiotic inactivating enzymes that are carried on transposons.

Two mechanisms for transposition:

1. Cut and paste - leaves a hole in the donor DNA
2. Replicative

Some transposons can do both types of transposition.

Question 2

Cut and paste

Answer 2

To initiate recombination, the transposase binds to the terminal IRs. It then brings the two ends of the transposon DNA together to form a stable protein-DNA complex which is a dimer or a tetramer of the transposase (called the transposome).
Then the transposase cleaves the DNA strand at each end of the trasnsposon at the flanking host DNA region.
The free 3’OH ends of the transposon DNA attack the target DNA phosphodiester bonds.
The DNA joining reaction occurs by a one step transesterification reaction called DNA strand transfer (similar to the reaction involved in RNA splicing).
Integration is done by DNA repair enzymes and DNA ligase.
Cut and paste also leaves a double strand break in the donor DNA which is repaired through homologous recombination.

Question 3

Replicative

Answer 3

As before, the transposome assembles on the donor DNA. The cleavage is achieved by the transposome nicking the DNA at each end of the two junctions between the transposome and the flanking host DNA. This cleavage liberates the 3’OH DNA ends on the transposon sequence, though unlike cut and paste, the transposon DNA is not excised.
The 3’OH ends are then joined to the target DNA site by the DNA STRAND TRANSFER REACTION. The intermediate generated is a doubly branched DNA molecule where the 3’ ends are attached to the new target site and the 5’ ends remain joined to the old flanking DNA.
The intermediate has the structure of a replication fork. The 3’ OH end in the cleaved target DNA serves as a primer for DNA synthesis. Replication proceeds through the transposon sequence and stops at the second fork. This replication produces two copies of the transposon DNA.