Holliday intermediates are associated with both homologous genetic recombination and some site-specific recombination systems. How does their formation differ in the two types of reactions?

Compare and contrast the DNA sequence requirements of homologous genetic recombination, site-specific recombination, and transposition.

If two FRT sites are present in a single DNA molecule in the same orientation, the Flp recombinase will promote a deletion of the DNA between the two sites. If the FRT sites are altered so that their core regions have a perfectly symmetric (palindromic) sequence instead of the usual asymmetric sequence, how might the fate of the intervening DNA in the reaction change? (Ignore any intermolecular reactions between the DNA molecules.)

When a site-specific recombinase brings two recombination sites together to form an initial complex for reaction, how many separate recombinase active sites are present in the complex? How many of those active sites are conformationally set up to catalyze a reaction?

Draw the products of Cre-lox–mediated site-specific recombination reactions for the recombination target sites and orientations (indicated by arrows) in the illustrations below.

What are the possible products of Flp recombinase–mediated site-specific recombination for the DNA molecule shown below? Arrowheads indicate the location and orientation of FRT sites. X and Y denote altered FRT sites that are functional but will not cross-react with each other. Draw all the products possible if recombination occurs at (a) the X sites and (b) the Y sites, and if two recombination events occur at (c) the X sites and then the Y sites, and (d) the Y sites and then the X sites.

516

Which of the following DNA molecules are appropriate substrates for the Hin recombinase? Arrowheads indicate the location and orientation of hix sites.

The bacterial transposon Tn3 uses a replicative transposition pathway to move from one DNA molecule to another. Tn3 encodes not only a transposase but a site-specific recombinase. Suggest a role in transposition for the site-specific recombinase in this system.

The Flp and Cre-lox site-specific recombination systems are widely used in biotechnology to promote genomic rearrangements in eukaryotic cells. The bacteriophage λ site-specific recombination system (see the How We Know section in this chapter) is rarely used for this purpose, even though it was the first such system discovered. Suggest why the λ system is not used.

Cut-and-paste transposition results in a short repeated sequence flanking the newly inserted transposon. The DNA sequence shown below is the target site for the transposition of a cut-and-paste transposon. Indicate with arrows the phosphodiester bonds targeted for transesterification by the transposon 3′ ends to generate a 5 bp repeat, 5′-AGGCT-3′, at both ends of the newly inserted transposon.

Many retroviruses have sequences complementary to the 3′ end of one of the tRNA molecules prominent in host cells infected by the virus. What is the purpose of this complementarity?

The transposase from the bacterial transposon Tn5 initiates transposition by creating a double-strand break at each end of the transposable element. How many transposase active sites are needed to generate one double-strand break, and how many phosphoryl transfers are catalyzed in that process?

Many TP (non-LTR) retrotransposons encode a reverse transcriptase that also has endonuclease activity. What is the primary function of the endonuclease activity?

The human genome has more than a million copies of the SINE element Alu. These transposons are found in the DNA between genes, and often in the introns of genes, but very rarely in gene exons. Explain why this is so.