Barabas Group
Mechanism of DNA recombination and its applications for research and therapy
Figure 2. The structure of the IS608 transpososome, modeled based on a series of crystal structures. Barabas et al, Cell 2008
Previous and current research
Controlled DNA rearrangements are essential for survival on all levels of life from individual cells to populations. Our lab is interested in understanding how DNA recombination is carried out on the molecular and cellular levels. We mainly focus on DNA transposons, a class of mobile genetic elements, which 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 necessary DNA cleavage and joining reactions. Transposons can be engineered to carry desired genetic information, and off er stable and heritable modifications of a target genome. Consequently, these ‘jumping’ DNA 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 their molecular mechanism of transfer, target site selection and cellular control. We currently study: i) the movement of various DNA transposons; and ii) RNA-based regulatory pathways that control the efficiency of transposition. Our techniques include structural biology (mainly X-ray crystallography), molecular biology, biochemistry and cell culture assays.
The reactivated transposon Sleeping Beauty has recently become a favoured genetic tool used for forward mutagenesis screens, identification of oncogenes, mapping regulatory landscapes, chromosomal engineering (Ivics et al, 2009) and even in gene therapy clinical trials (Williams, 2008). However, the mechanism of SB transposition is poorly understood. Our on-going structural and functional studies will off er a mechanistic understanding and invaluable insights for rational design of transposition cassettes.
Site-specic elements: One of the main obstacles of gene therapy is integration of the therapeutic gene at unwanted genomic locations. Site-specific recombinases may offer a solution. Our recent work revealed the mechanism of the bacterial Insertion Sequence, IS608, that integrates site-specifically at short DNA sequences (Barabas et al, 2008). We found that target site recognition is achieved by the transposon DNA base pairing with target DNA. The site of insertion can be easily altered by changing a few nucleotides in the transposon (Guynet et al, 2009). We are currently investigating if this target recognition strategy can be expanded to target potentially unique genomic sites. We are also studying a newly discovered mobile element, the plasticity zone transposon (TnPZ) from Helicobacter pylori (Kersulyte et al, 2009). This element shares features with Xer recombinases and may move via bacterial conjugation. We want to learn how this transposon moves and selects its 7nt long specific target site sequence.
In eukaryotes transposon silencing is mainly achieved by regulatory piRNAs (PIWI-interacting RNAs). They facilitate transposon silencing by triggering both transposon DNA methylation and mRNA degradation by cytoplasmic endonucleases. We investigate various proteins involved in this pathway to reveal their role and mechanism of action.
Future Projects and Goals
- To understand transposition in the cellular context, we will identify host proteins that interact with the transposase protein or transposon DNA. The eff ect of host factors on transposition efficiency will also be analysed.
- We will select representative targets from several transposon and recombinase families to capture a broader picture of recombination pathways applied by nature.