Mechanism of mobile DNA elements and their applications for research and therapy
Figure 1: The structure of the IS608 transpososome, modelled based on a series of crystal structures
Figure 2: Crystal structure of the primary piRNA biogenesis factor Zucchini reveals its endonuclease function
The Barabas group uses structural and molecular biology approaches to investigate how controlled DNA rearrangements are carried out and regulated.
Previous and current research
Our research focuses on DNA transposons, a class of mobile genetic elements that can autonomously move from one genomic location to another. They contain specific DNA sequences at their ends and encode a transposase enzyme that catalyses all the required DNA cleavage and joining reactions. Transposons can be engineered to carry desired genetic information, and can stably and heritably modify a target genome. Thus, these ‘jumping’ elements offer attractive tools for genetics and human gene therapy. To support the future development of transposon-based genetic tools, we study their mechanism of movement. We strive to understand the structure and assembly process of functional complexes, the chemistry they use to cut and paste DNA, as well as their target-site selection and regulation in the cell. Our techniques include structural biology (mainly X-ray crystallography), molecular biology, biochemistry, biophysics and cell-based assays. We currently study: i) the movement of various DNA transposons; and ii) RNA-based regulatory pathways that control transposition.
Sleeping Beauty: This reactivated transposon has recently become a favoured genetic tool for forward mutagenesis screens, mapping gene regulatory landscapes, chromosomal engineering and even gene therapy (Ivics et al., 2009). We perform structural and functional studies to obtain a mechanistic understanding of this transposon and, in collaboration with the Gavin group, we also investigate how it interacts with human cells.
Sequence-specific elements: One of the main obstacles of gene therapy is integration of the therapeutic gene at unwanted locations. Therefore, we seek tools that integrate a genetic cargo to selected specific sequences and can provide a solution. Our recent work revealed the mechanism of the bacterial Insertion Sequence IS608, which uses a short sequence in the transposon DNA to guide its integration to a specific sequence via base pairing (Barabas et al., 2008). Consequently, the site of insertion can be altered by making point mutations in the transposon (Guynet et al., 2009). We are investigating if this target recognition can be extended to target unique genomic sites, which may provide an easy to customise programmable genetic tool. We are also studying a newly found mobile element – the plasticity zone transposon in Helicobacter pylori (Kersulyte et al., 2009) – focusing on how it moves and integrates to a 7nt-long specific sequence.
Transposon regulation: To avoid deleterious outcomes, cells must keep their transposons under control. Small RNAs can control transposon activity in various ways. In collaboration with the Carlomagno and Pillai groups, we investigate these processes in prokaryotes and eukaryotes. Our recent work on the eukaryotic piRNA pathway has revealed the structure and function of a novel component, a piRNA biogenesis factor called Zucchini.
Future Projects and Goals
- Building on mechanistic insights, we will develop novel genetic tools for genomic screening and transgenesis.
- Exploring the potential of transposon-based genetic tools in synthetic biology.
- Investigating the molecular mechanisms of somatic genome assembly in ciliated protozoa (a large-scale DNA rearrangement process resembling transposition).