Transposition : a new family of prokaryotic transposons – Mobigene

Scientific background and objectives: The project is designed to determine the transposition mechanism of the IS200/IS605 group of transposable elements. This newly recognised class is present throughout the Bacteria and Archaea and in some eukaryote giant DNA viruses (e.g. Mimi and Phycodnaviruses), often in high numbers. They may also substitute for homing endonucleases in type I bacterial introns. Their transposases (TnpA; the enzyme necessary for DNA mobility) resemble enzymes used in conjugal plasmid transfer, bacteriophage and plasmid rolling circle replication and in the replication of eukaryotic viruses (e.g. AAV and TYLCV). These ISs therefore represent an important evolutionary crossroad and their wide distribution argues for a significant role in genome plasticity. We have shown that one member, IS608, excises from the donor molecule as a circle; that the donor backbone is precisely resealed; that there is a correlation between excision and transposition suggesting that the excised transposon circle is an intermediate in the transposition pathway; and that a 5' flanking target 5'TTAC is essential for integration, excision and transposition. Transposition likely occurs using a ssDNA intermediate. Moreover, transposition of a second member, ISDra2, from D. radiodurans, is induced following irradiation, a process which generates ss DNA. Description of project, methodology: For IS608 we will study: TnpA DNA interactions, binding dynamics and catalytic properties. IS608 ends are unusual and include extensive secondary structures. TnpA binds each ds end but shows high affinity for the 'top' strand. It cleaves ss oligonucleotides forming a covalent 5' P-Tyr bond. We will use both in vivo and in vitro approaches comprising genetic, biochemical and fluorescent-based methods, to investigate the sequence/structure elements necessary for DNA recognition, substrate (transposon ends and target DNA) binding, cleavage and recombination. TnpA forms a dimer in which each monomer contributes to the two shared catalytic sites and the two shared DNA binding sites involving residues from each monomer. In the absence of DNA, the active site is in a non-functional configuration. Binding of appropriate DNA activates the enzyme. We will probe the inherent secondary DNA structure of the ends with and without TnpA; develop an in vitro transposition system, building on our experience with other, more traditional systems, and we will construct in vivo assays for screening transposition activity to use both for screening TnpA- and host factor-mutants obtained by random or site directed mutagenesis. To provide a broader picture of IS200/IS605 family transposition, we will also study ISDra2 transposition both in vitro and in vivo. In vivo studies will address irradiation-induced transposition and, using a transposon-mediated mutant library and a reporter system specific for excision and for insertion, we will explore host factors involved in ISDra2 transposition. Similar studies will be carried out for IS608 using an analogous reporter system. The approaches used in the project include biochemistry, genetics and biophysics. Expected results: The results obtained will include a detailed description of the transposition of this widely distributed class of mobile genetic element, its regulation, and its interaction with host factors and therefore host cell physiology. In addition, we expect to use this experimental system to enhance the low energy CD methodology developed in the Johnson lab (IPBS, Toulouse). Coupled with ongoing structural studies undertaken in collaboration with the Dyda lab (NIH, USA) these experiments will reveal further interesting mechanistic insights and principles in addition to those already uncovered in this system such as DNA-induced conformational changes in TnpA, the ability to change metal ion specificity for the reactions.