Functional analysis of the domain responsible for target specificity and development of new specificity on IS30 transposase

Functional analysis of the domain responsible for target specificity and development of new specificity on IS30 transposase

János Kiss PhD, Zita Nagy and Mónika Szabó

Agricultural Biotechnology Center,

Environmental Biosafety Research Institute, Gödöllő, Hungary

Co-partner: Prof. Michael Chandler

Laboratoire de Microbiologie et Genetique Moleculaire de CNRS, Toulouse, France

Insertion sequences play significant role in rearrangements of genetic material of living organisms, thus these elements are important objects and tools of genetic research. The transpositional mechanisms of the Escherichia coli element IS30 are well known – mainly due to our earlier works. Transposition of IS30 occurs via an active intermediate structure composed of two directly repeated elements. Formation of this intermediate and its integration into the target DNA is catalysed by the transposase enzyme (Tpase) encoded by the element. Main domain structure of the Tpase has also been described. The N-terminal part contains special DNA-binding regions, while C-terminal part is responsible for intermediate formation. The IS30 transposition shows expressed sequence-specificity in target selection, which is an additional function of the enzyme.

In the course of this grant we are going to localise and characterise the DNA-binding regions of the Tpase that are responsible for recognition of inverted repeats of the element and the target DNA. Our hope is to get deeper insight into the functional organisation of the enzyme through comparative analysis of the IS30-related Tpases and mutational analysis of IS30 Tpase. The main goal of this work is the determination of regions active in targetspecificity function of the enzyme. To achieve these aims we are going to apply computer analysis, directed mutagenesis and other microbial and biochemical methods.

Importance of the project is getting more detailed knowledge on a transposase enzyme. This may help in development of an IS30-based widely applicable gene targeting and mutagenesis system, which can be a useful tool in functional genomics and applied biotechnology, as well.

Supported by the Hungarian Academy of Sciences OTKA F038204