Barabas Group
Mechanism of DNA recombination and its applications for research and therapy
Figure 1. Ex vivo gene therapy, an alternative, perhaps safer route of gene therapy, that allows application of transposon based delivery vehicles.
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 play a key role in various biological pathways, like DNA repair, adaptive immunity and evolution; hence they are essential for survival on all levels of life from individual cells to populations. Our lab is interested in how DNA recombination is carried out by various systems. We are particularly interested in DNA transposons because these mobile genetic elements offer attractive genetic tools. They are currently starting to be applied as gene delivery vehicles in mammalian functional genomics and human gene therapy. To support the development of transposon based genetic tools, we study their mechanism of movement. Our techniques include structural biology (mainly X-ray crystallography), molecular biology, biochemistry and cell culture assays.
Gene therapy is a promising strategy for many diseases that challenge modern medicine. Its objective is to correct or complement faulty genetic information with therapeutic genes. However, there are still major problems to overcome before gene therapy can become a common medical technique. For example, there is a pressing need for gene delivery vehicles that provide safe and efficient integration of the therapeutic gene into the host chromosome and allow long term expression without causing harmful DNA rearrangements.
DNA transposons are mobile DNA elements that have the ability to move from one genomic location to another. They contain specific DNA sequences at their ends, and encode a transposase enzyme that catalyzes all necessary DNA cleavage and joining reactions. Transposons can also be engineered to carry desired genetic information, offering stable and heritable modifications of a target genome. Therefore, we want to better understand the mechanism of transfer, target site selection and cellular control of various DNA transposition systems on molecular and cellular levels. Two major current interests include i) the Sleeping Beauty transposon system and ii) site specific bacterial transposons.
Sleeping Beauty was reconstructed from fish genomes. Due to its relatively high transposition efficiency in human cells, SB has been successfully applied to identify oncogenes and tumor suppressors (Dupuy et al, 2005), and to characterize unknown genes. More recently, SB is being applied in a gene therapy clinical trial (Williams, 2008). However, the mechanism of SB transposition and its interactions with the host have yet to be unraveled. Our on-going structural and in vitro functional studies shall offer a mechanistic understanding and invaluable insights for rational design of transposition cassettes.
Site-specific elements: One of the main obstacles of gene therapy is the occurrence of harmful genome perturbations due to integration at unwanted 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. We found that the transposase co-opts a segment of the transposon DNA to be part of its active site, and uses it to recognize the site of insertion (Barabas et al, 2008). A consequence of the observed target site recognition strategy is that the site of insertion can be easily altered by changing a few nucleotides in the transposon Guynet et al, 2009). This DNA based recognition might also be expandable with the aim of targeting longer potentially unique genomic sites. We are currently investigating this possibility.
We are also studying a newly discovered site specific mobile element, the so called plasticity zone transposon (TnPZ) from Helicobacter pylori (Kersulyte et al, 2009). This mobile element appears to move with a mechanism similar to bacterial conjugation and integrates to 7nt long specific sites. We are curious to find out how this transposon moves and selects its target site.
Future Projects and Goals
- We will initiate structural and mechanistic studies with the site-specific eukaryotic transposons, the Helitrons.
- We will select representative targets from several transposon and recombinase families to capture a broader picture of the alternative recombination pathways applied by nature.
- In general, we are fascinated by the enormous information content of DNA and the sophisticated machinery maintaining, interpreting and manipulating this code. On the long term, we intend to broaden the range of our projects in these directions.