Tn3 resolvase

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Tn3 resolvase, its res site, and the reaction that it catalyses

Tn3 resolvase or is one out of three proteins that are encoded on the 4957 bp Tn3 transposon. Apart from Tn3 resolvase or tnpR, this transposon also carries a transposase or tnpA and a β-lactamase Bla that confers resistance to β-lactam antibiotics. Initially discovered as a repressor of transposase, resolvase also plays a role in facilitating Tn3 replication (Sherratt 1989). Tn3 replicates by intermolecular integrative transposition. This process is catalysed by the Tn3 transposase and it results in a fusion of the original host DNA with the target DNA molecule creating a “cointegrate” along with the replication of the transposon. To separate the host and target molecules Tn3 resolvase executes site-specific recombination between the old and new copy of transposon at a speciffic site called res, which is present in each copy of the transposon. Res is 114 bp long and it consists of 3 sub-sites, namely sites I, II and III. Each of these sites is of different lengths (28, 34 and 25bp, respectively) and they are unevenly spaced with 22bp separating sites I and II and only 5bp between sites II and III. The sites consist of 6bp inverted repeat motifs flanking a central sequence of variable length. These motifs act as binding sites for resolvase, so that each site binds a resolvase dimer but with varying affinity and probably a slightly different protein-DNA complex architecture (Abdel-Meguid 1984; Blake et al., 1995). All three sub-sites are essential for recombination. At recombination, two directly repeated res sites with resolvase dimers bound to each sub-site, come together to form a large complex structure called the synaptosome. Resolvase bound to sites II and III initiates the assembly of this complex. In this structure, exact architecture of which is still unclear, two res sites are intertwined in such a way as to juxtapose two copies of site I, allowing resolvase dimers bound to each site to form a tetramer. Again, it is the interaction between the resolvase dimers bound at accessory sites (sites II and III) and resolvase at site I that causes the two dimers to synapse and form a tetramer. After the tetramer is formed it becomes activated and the top and bottom DNA strands are simultaneously cleaved in the middle of the site I with a 2bp overhang. The strand exchange ensues by as yet unknown mechanism with a resulting net rotation of 180°. The strand exchange is then followed by the religation (Stark et al., 1992). Recombination between two directly repeated res sites separates, or resolves, the “cointegrate” into two original molecules, each one now containing a copy of the Tn3 transposon. After resolution these two molecules remain linked as a simple two-noded catenane which can be easily separated in vivo by a type II topoisomerase (Grindley 2002). Wild type resolvase system absolutely requires a supercoiled substrate and that the recombination sites are oriented in a direct repeat on the same DNA molecule. However, a number of “deregulated” or “hyperactive” mutants that have lost the requirement for the accessory sites have been isolated. These mutants are capable of catalysing recombination between two copies of site I only, which basically reduces the recombination site size from 114bp to only 28bp (Arnold et al., 1999; Burke et al., 2004). Further more these mutants have no supercoiling or connectivity requirements (Arnold et al., 1999) and have been shown to work in mammalian cells (Schwikardi and Droge, 2000). Hyperactive resolvase mutants have so far proven useful in creating resolvases with altered sequence specificity (Akopian et al., 2003) but also in structural work (Li et al., 2005).

The entire resolvase recombination reaction can be reproduced in vitro, requiring only resolvase, a substrate DNA and multivalent cations, using either wild type protein or hyperactive mutants (Reed 1981; Arnold et al., 1999).

Hyperactive resolvase mutants, if further developed, could become an alternative to Cre and FLP, the most commonly used recombination systems in molecular biology to date.

[edit] References

1.Sherratt, D. J. (1989). Tn3 and related transposable elements: site-specific recombination and transposition. In Berg, D. E., Howe, M. (eds) Mobile DNA. American Society for Microbiology, Washinghton, DC pp. 163-184

2. Abdel-Meguid, S.S., Grindley, N.D., Templeton, N.S. and Steitz, T.A. (1984). Cleavage of the site-specific recombination protein gamma delta resolvase: the smaller of two fragments binds DNA specifically. Proc. Natl. Acad. Sci. USA. 81, 2001-2005

3.Blake, D. G., Boocock, M. R., Sherratt, D.J. and Stark, W. M. (1995). Cooperative binding of Tn3 resolvase monomers to a functionally asymmetric binding site. Curr. Biol. 5, 1036-1046 4.Stark, W.M., Boocock M.R. and Sherratt, D.J. (1992). Catalysis by site-specific recombinases. Trends Genet. 8, 432-439.

4.Grindley, N.D.F. (2002). The movement of Tn3-like elements: transposition and cointegrate resolution. In Mobile DNA II, Craig, N., Craigie, R., Gellert, M. and Lambowitz, A. (ed.), pp272-302. ASM Press, Washington, DC, USA

5.Arnold, P.H., Blake, D.G., Grindley, N.D.F., Boocock, M.R. and Stark, W.M. (1999). Mutants of Tn3 resolvase which do not require accessory binding sites for recombination activity. EMBO J. 18, 1407-1414

6.Burke, M.E., Arnold, P.H., He, J., Wenwieser, S.V.C.T., Rowland, S-J., Boocock, M.R., and Stark, W.M. (2004). Activating mutations of Tn3 resolvase marking interfaces important in recombination catalysis and its regulation. Mol. Microbiol. 51, 937-948.

7.Schwikardi, M. and Droge, P. (2000). Site-specific recombination in mammalian cells catalysed by γδ resolvase mutants: implications for the topology of episomal DNA. FEBS Letters 471, 147-150

8.Akopian, A., He, J., Boocock, M.R. and Stark, W.M. (2003). Chimeric recombinases with designed DNA sequence recognition. Proc. Natl. Acad. Sci. USA 100, 8688-8691

9.Li, W., Kamtekar, S., Xiong, Y., Sarkis, G.J., Grindley, N.D.F. and Steitz, T.A. (2005). Structure of a synaptic γδ resolvase tetramer covalently linked to two cleaved DNAs. Science 309, 1210-1215.

10.Reed, R. R. and Grindley, N. D. (1981). Transposon-mediated site-specific recombination in vitro: DNA cleavage and protein-DNA linkage at the recombination site. Cell. 25, 721-728