Chapter 14

Question 14.1

In tyrosine-class site-specific recombination systems, the Holliday intermediate is generated in a precise set of cleavage and strand-transfer steps, all occurring at a single, unique DNA sequence. In homologous recombination, Holliday intermediates can appear at any sequence and are generated by the strand invasion and branch migration promoted by RecA recombinases.

Question 14.2

Homologous recombination occurs only where two DNA molecules have identical or very similar sequences over a significant region; any sequence is permitted. Site-specific recombination occurs only at particular DNA sequences that are recognized, bound, and recombined by the recombinases. Transposition, with a few exceptions, can occur at almost any sequence.

Question 14.3

The DNA between the sites would undergo both deletions and inversions.

Question 14.4

Four active sites are present; only two are in the conformation required to catalyze a reaction.

Question 14.5

Question 14.6

  1. Recombination at the X sites inverts the intervening sequence because the X sites are in opposite orientations.

  2. Recombination at the Y sites deletes the intervening sequence because the Y sites are in the same orientation, leaving only one copy of the X site and one of the Y site. (c) If the X sites react first, the orientation of one Y site changes, leading to an inversion in the later reaction with the Y sites. (d) If the Y sites react first, deletion occurs. Reaction of the (deleted) X site on the circle with the (remaining) X site on the original DNA yields the same product as in (c).

Question 14.7

Only (b) is an appropriate substrate. The Hin-hix system works only when the two hix sites are in the opposite orientation and on the same supercoiled DNA molecule.

Question 14.8

Replicative transposition generates a cointegrate intermediate that joins the donor and target DNAs together. The cointegrate is resolved (the DNAs are separated) by the transposon-encoded site-specific recombinase (the Tn3 resolvase).

Question 14.9

The λ site-specific system requires additional proteins, besides the Int recombinase, to promote the reaction. To adapt this system to eukaryotic cells, these additional proteins, as well as the recombinase, would have to be expressed. The λ attP site is also more complex, and is a larger segment of DNA to clone, than the FRT and lox sites.

Question 14.10

Question 14.11

The tRNA, carried by the virus from one host to another, provides the primer for DNA synthesis by reverse transcriptase.

Question 14.12

Only one active site is required to create one of the double-strand breaks. Three phosphoryl group transfer reactions are catalyzed in succession to bring this about.

Question 14.13

The reverse transcriptase of TP (non-LTR) retrotransposons makes use of a 3′ end derived from the target DNA to prime viral DNA synthesis. The endonuclease cleaves a phosphodiester bond in the target, exposing the needed primer terminus.

Question 14.14

Exons are the coding regions of genes. Insertion of a transposon of any type into an exon would almost certainly disrupt the activity of the protein encoded by the gene.

Question 14.15

(a) If Cre were used for the insertion, the cassette itself could be altered during the insertion process. (b) When the Flp reaction is complete, intact FRT sites flank the cassette. Each FRT could be a target for a new insertion event. (c) The core sequence (see Figure 14-2a) must be modified to prevent recombination between the different Cre sites. (d) Using R, Y, and C to denote RFP, YFP, and CFP, the possible combinations are: RRR, YYY, CCC, RRY, RRC, RYY, RCC, YCC, YYC, and RYC.