The role of MuB in selecting transposition targets of bacteriophage

Phage Mu transposes promiscuously, employing MuB protein for target capture. MuB forms stable filaments on A/T-rich DNA, and a correlation between preferred MuB binding and Mu integration has been observed. We have investigated the relationship between MuB-binding and Mu insertion into ‘hot’ and ‘cold’ Mu targets within the E. coli genome. Although higher binding of MuB to select hot versus cold genes was seen in vivo, the hot genes had an average A/T content and were less preferred targets in vitro, whereas cold genes had higher A/T values and were more efficient targets in vitro. These data suggest that A/T-rich regions are unavailable for MuB binding, and that A/T content is not a good predictor of Mu behavior in vivo. Insertion patterns within two hot genes in vivo could be superimposed on those obtained in vitro in reactions employing purified MuA transposase and MuB, ruling out the contribution of a special DNA structure or additional host factors to the hot behavior of these genes. While A/T-rich DNA is a preferred target in vitro, a fragment made up exclusively of A/T was an extremely poor target. A continuous MuB filament assembled along the A/T region likely protects it against the action of MuA. Our results suggest that MuB binds E. coli DNA in an interspersed manner utilizing local A/T richness, and facilitates capture of these bound regions by the transpososome. Actual integration events are then directed to sites that are in proximity to MuB filaments but are themselves free of MuB. 47 Introduction Transposable elements employ a variety of strategies for selecting target sites, and display a wide spectrum of target specificities (Craig, 1997; Sandmeyer, 1998). The transposases of some elements choose target sites directly, while others use accessory proteins to mediate this choice. The sequence, structure, as well as transcription and replication status of DNA can influence insertion preference of different elements. The study of target site selectivity provides insights not only into transposition mechanisms, but genome structure and function as well. Phage Mu is an extremely efficient transposon which gets its name ‘mutator’ from its ability to insert essentially randomly within the E. coli chromosome (Taylor, 1963), a randomness confirmed in early studies by fine-mapping of Mu insertions within a single gene (Bukhari, 1972). Later studies, however, showed preferential regions for Mu insertions within a plasmid (Castilho and Casadaban, 1991), near the control region of some genes (Manna et al., 2001; Wang and Higgins, 1994), as well as in the whole E. coli genome (Manna et al., 2004; Manna et al., 2005). The preferential integration observed within a plasmid was correlated to binding of the accessory protein MuB (Mizuuchi and Mizuuchi, 1993). DNaseI footprinting identified a MuB-protected region on the plasmid, and Mu insertions were seen to occur on either side of this protected region. A target sequence consensus of 5'-NY(G/C)RN-3' was identified, which was shown to be independent of the presence of MuB i.e reflected the preference of the transposase (Mizuuchi and Mizuuchi, 1993). A more detailed in vitro analysis of this target site