The mechanism of DNA cleavage in Hsmar1, a mariner-family transposon from humans. (360G-Wellcome-083542_Z_07_Z)

The mariner transposons are promising tools for the genetic manipulation of mammals. However, their precise mechanism remains the largest gap in our fundamental understanding of the DNA-based transposons. Progress on both fronts has been hindered by the unique properties of the different transposons. The most efficient system in vertebrates, has proven recalcitrant in vitro. Conversely, those elements active in vitro, perform poorly in mammalian cells. We propose to break this impasse using Hsmar1. Hsmar1 is highly amenable in vitro, and is presumably adapted to its mammalian host. The efficiency of mariner transposition appears to be limited by the stability and/or aggregation of the synaptic complex. We will investigate changes in the arrangement and stoichiometry of the subunits during assembly of the synaptic complex and intermediate stages of the reaction. This will address weather different subunits perform different functions, and whether this is dictated by their stru ctural placement within the complex. During this work we will generate mutations to improve solubility, transposon end binding and resistance to aggregation. Combined with improved physical methods for preparing the complex, this will facilitate structural studies. An atomic structure for the complex will permit direct engineering of the system for a number of practical applications.