The Mu Transposase Interwraps Distant DNA Sites within a Functional Transpososome in the Absence of DNA Supercoiling

Abstract

A Mu transpososome assembled on negatively supercoiled DNA traps five supercoils by intertwining the left (L) and right (R) ends of Mu with an enhancer element (E). To investigate the contribution of DNA supercoiling to this elaborate synapse in which E and L cross once, E and R twice, and L and R twice, we have analyzed DNA crossings in a transpososome assembled on nicked substrates under conditions that bypass the supercoiling requirement for transposition. We find that the transposase MuA can recreate an essentially similar topology on nicked substrates, interwrapping both E-R and L-R twice but being unable to generate the single E-L crossing. In addition, we deduce that the functional MuA tetramer must contribute to three of the four observed crossings and, thus, to restraining the enhancer within the complex. We discuss the contribution of both MuA and DNA supercoiling to the 5-noded Mu synapse built at the 3-way junction.Many recombinases, including the phage Mu transposase, are strictly dependent on DNA supercoiling for activity. These recombinases and their accessory factors take advantage of both the conformational and thermodynamic properties of supercoiled DNA, which include DNA unwinding, formation of single-stranded regions, extrusion of cruciform structures, sequence-dependent induction of Z-DNA conformation, and DNA bending as well as an increase in the local concentration of DNA sites (1). The present study was undertaken to examine the role of DNA supercoiling in establishing the five-noded topology observed in Mu transposition complexes (2, 3).The Mu transposition pathway is shown in Fig. 1 (4). Two families of DNA sites (high affinity attL and attR sites and lower affinity enhancer (E) 1 sites (Fig. 1A)) are recognized by the transposase MuA through separate DNA binding domains. On the linear phage genome, attL and attR are 37 kilobases apart, whereas the enhancer is 1 kilobase from attL (5). MuA monomers likely first bind the att ends and then interact with the enhancer through bridging contacts. A criss-crossed network of att-enhancer interactions has been deduced (6, 7), an ordered progression of which is thought to result in the series of complexes observed during transposition (Fig. 1B).Mu transposition is strictly dependent on DNA supercoiling of the donor substrate under normal reaction conditions. Several roles for supercoiling have been deciphered. Supercoiling increases the binding affinity of MuA for the L and R ends (8) and of the accessory host factor HU for the L end (9), favors bending at the enhancer (10), facilitates interwrapping of the L, E, and R segments within the Mu synapse (11, 2), and assists "open termini" formation, a rate-limiting step in the assembly of the transpososome (12-16). Supercoiling is not required for the chemical steps of transposition per se (4).During assembly of the five-noded Mu synapse, the enhancer first uniquely interacts with R and interwraps with it twice to form an ER complex (Fig. 1B) (2, 3). In the absence of Escherichia coli HU protein, L i...

Determinants of Product Topology in a Hybrid Cre-Tn3 Resolvase Site-specific Recombination System

“…13,[19][20][21][22][23][24] The results presented here are relevant to the analysis of these other systems. In particular, we conclude that the effects of recombination crossover site orientation, the exact length of spacer DNA, and DNA supercoiling must be taken into account when using these systems to deduce synapse structure.…”

Enhancer-independent Mu transposition from two topologically distinct synapses

“…The contribution of the Mu ends themselves to the topological specificity underscores a similar logic. Finally, the enhancer must be involved in yet another role in transposition, because it stays sequestered within the transpososome even after strand transfer is complete (8,18).…”

“…The overall topology of this synapse is maintained throughout transposition (8). The transposase MuA can recreate a similar topology on nicked substrates in the presence of Me 2 SO, suggesting that formation of the interwrapped architecture is an active process rather than a fortuitious trapping of the DNA crossings already present in supercoiled DNA (18). Although the dual DNA-binding specificity of MuA for sites within the ends as well as the enhancer likely sets up the E-R͞E-L crossings, the L-R crossings must be brought about by a different set of interactions.…”

Determining the topology of stable protein–DNA complexes

“…In a later study, difference topology experiments were used to investigate the role of supercoiling [41]. Many proteins require DNA supercoiling for binding and/or activity.…”