Visualizing Mu transposition: assembling the puzzle pieces.

Mobile genetic elements, once a curiosity of maize genetics, are now known to be remarkably prevalent in the genomes of organisms ranging from pathogenic bacteria to humans. The varied consequences of these elements include the alteration of gene expression and the spread of drug resistance genes among bacteria. Additionally, many viruses and bacteriophages that covalently integrate their own genome into their hosts’ do so by mechanisms similar to those used by more domesticated mobile elements that do not normally leave the cell (for review, see Curcio and Derbyshire 2003). Bacteriophage Mu was the first transposition system studied in vitro, and has in many ways served as a paradigm for understanding the mechanisms of mobile DNA elements (Chaconas and Harshey 2002). In this issue of Genes & Development, Chaconas, Ottensmeyer, and colleagues (Yuan et al. 2005) present an electron microscopy-based 3D model of the protein–DNA complex responsible for Mu transposition (the transpososome). This model not only ties together years of accumulated biochemical and structural data, but also provides interesting new insights. Mu is a member of the first characterized and perhaps most well studied family of mobile elements, often referred to as the “classical DNA transposons” or the “DDE transposons.” The latter name reflects the conserved active sites of the element-encoded transposase enzymes, which contain three carboxylate side chains that bind catalytic Mg ions (Mizuuchi and Baker 2002). This large family includes the bacterial transposons Tn5, Tn7, and Tn10 as well as phage Mu, which is covalently inserted into the host genome and, in lytic phase, replicates via a massive burst of transposition. The family can also be extended to include retroviruses such as HIV, whose integration is catalyzed by an enzyme closely related to the DDE transposases. A generalized pathway for the transposition of these elements is shown in Figure 1. The first step is simple hydrolysis of the DNA backbone at the 3 ends of the element. This is reflected in the structural similarity between the active site-containing domains of these transposases and that of RNAse H, which hydrolyzes the RNA strand of RNA–DNA hybrids (Rice and Baker 2001). However, unlike nucleases that use only water as a nucleophile, transposases can also use the 3 hydroxyls released in the first step to attack another DNA segment (the “target”). This strand transfer reaction is rather odd in that there is no obvious chemical driving force (the total number of phosphodiester bonds is unchanged). It appears to be driven forward solely by product binding energy. As a consequence these “enzymes” do not usually turn over, and in the Mu case the final complex is known to be so stable that it blocks replication until it is removed in an ATP-dependent fashion by ClpX (Nakai et al. 2001). The fate of the second DNA strand of the original duplex varies. In replicative transposition systems such as Mu it remains intact, and the resulting branched DNA is later converted to a replication fork. In Tn7 transposition, it is cleaved by a second nuclease (Sarnovsky et al. 1996), whereas in Tn10 and Tn5 transposition it is cleaved by successive hairpinning and hydrolysis reactions catalyzed by the same active site as the one that mediates the initial cleavage (Kennedy et al. 1998; Bhasin et al. 1999). The latter pathway bears striking resemblance to the series of reactions carried out by the RAG proteins during immunoglobulin gene assembly in higher organisms (Jones and Gellert 2004). Transposition takes place within a large protein–DNA complex (termed a transpososome) that often includes additional regulatory features. For instance, it is important to the health of the transposon that the catalytic events at its two ends be coordinated. Biochemical experiments on Mu followed by structural studies of Tn5 transposase showed that this can be enforced by catalysis in trans (Aldaz et al. 1996; Savilahti and Mizuuchi 1996; Davies et al. 2000). Tight, specific binding of the transposase to the sequences near the transposon ends is accomplished not by the active-site domain itself but by one or more additional DNA-binding domains. Within the transpososome, the protomer that is bound specifically to one transposon end catalyzes the chemical events at the other end, and vice versa. Mu is a system particularly rich in regulatory features: For example, transpososome assembly is stimulated by an enhancer sequence found within the Mu genome that is bound by a separate domain of the transposase (MuA Correspondence. E-MAIL [email protected]; FAX (773) 702-0439. Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/ gad.1309305.