RUI: Structure/Function Analysis of the Himar1 Transposon

Duquesne University, Pittsburgh PA

Investigators

Abstract

The long-term goals of this project are to understand the structure and function of the Himar1 transposase. A series of in vitro and in vivo E. coli assays and screens will be used to determine the amino acids responsible for mediating the several activities of Himar1 transposase that constitute transposition. In vitro mutagenesis will be used to create a series of C-terminal truncations of transposase each of which will be screened by gel shift assays to determine how much of the protein can be deleted before DNA binding activity is eliminated. Random and specific point mutations in the DNA binding domain will then be introduced and the mutations assayed for their effects in vivo and in vitro. The boundaries of the catalytic domain, the part of the protein responsible for the DNA breakage and joining reactions, will be mutated randomly and at conserved positions based on an alignment of many mariner-family transposons and the mutants screened for the ability to either excise the transposon or insert it into a target. The motif responsible for mediating target site TA dinucleotide identification will be localized with an E. coli screen which will allow the identification of randomly mutated Himar1 transposase constructs that complete insertion into improper target sequences. Finally, the DNA sequence of the inverted terminal repeat (ITR) will be mutated to determine the minimum sequences required for transposition. High-resolution chemical footprinting will be carried out to determine which base pairs are specifically contacted by transposase. Finally, a random mutagenesis screen will be performed to determine the effects of each position in the ITR on transposition frequency. By understanding the structure and function of this transposable element it is hoped that its utility in a variety of species will be dramatically improved.