Homologous recombination is a fundamental cellular process. It rearranges genes both within and between chromosomes, promotes repair of damaged DNA and underpins replication. Recombination is characterised by the breakage and joining of DNA duplexes of identical or near-identical sequence, which may occur naturally during genetic exchange or in response to DNA damage. Double-strand DNA breaks are a particularly dangerous form of DNA damage, as they can lead to chromosomal aberrations. Human patients with defects in the normal cellular response to double-strand breaks, such as ataxia telangiectasia and Nijmegen breakage syndrome, are predisposed to cancer.
We are using genetics to understand how DNA recombination and repair operate in the archaea.
The archaea comprise the third domain of life, alongside bacteria and eukaryotes, and have only recently been discovered. They live in extremely harsh environments such as boiling acid pools or salt lakes, which pose enormous challenges for growth and DNA metabolism.
Double-strand break repair (DSBR) and synthesis-dependent strand annealing (SDSA) models of recombination
We are interested in how recombination has evolved to meet these challenges, and how it operates in a lineage that is fundamentally distinct from bacteria and eukaryotes.
Intriguingly, the archaea show many similarities to eukaryotic cells, particularly in the proteins used for DNA replication, repair and recombination.
Studies of the archaea may therefore generate stripped-down models that allow the dissection of more complex eukaryotic systems.
Tree of Life
Since most archaeal species are extremophilic and difficult to cultivate, current knowledge of recombination in the archaea is confined largely to comparative genomics and biochemistry.
We are developing a genetic and molecular systems using Haloferax volcanii as a model organism. Haloferax volcanii was isolated from the Dead Sea and is a halophile. It grows aerobically at 45°C in media containing 2.5 M NaCl, and can be cultivated in the laboratory with ease.
Haloferax volcanii cells growing on an agar plate and in liquid culture
Haloferax volcanii offers great potential for establishing tractable and informative genetic systems.
A number of auxotrophic mutants are available which in combination with antibiotic-resistance markers, an efficient transformation system, reporter genes, and plasmid vectors for cloning and gene expression provide a formidable arsenal of genetic tools.
Haloferax volcanii is one of only two archaeal species in which a natural process of genetic exchange has been observed. We are systematically deleting homologues of known recombination genes in Haloferax volcanii by "reverse genetics".
Genome sequence data is used to identify genes of interest, and deletions are made using the counter-selectable marker system.
Counter-selectable deletion system based on selection for uracil prototrophy and 5-FOA resistance
We are particularly interested in the radB gene. Archaea possess two RecA-like strand exchange proteins (RadA and B). A number of RecA homologues also exist in eukaryotes, and play distinct roles at different developmental stages. RadB is unique to the archaea, and has diverged significantly from its RadA counterpart. Unlike RadA, RadB does not promote DNA strand exchange.
A radA mutant of Haloferax volcanii has already been constructed, and displays recombination defects and sensitivity to DNA damage.
We have now made a radB deletion mutant of Haloferax volcanii, and preliminary results show that it is almost as sensitive to UV light as a radA mutant. This suggests that RadB plays an important role in DNA recombination and repair.
Haloferax volcanii radB mutants are sensitive to UV light
We are also making recombination mutants using "traditional" genetics. Random gene disruptions are generated in the Haloferax volcanii genome, and mutants screened for recombination defects using a blue/white colour assay.
Candidate mutants are assayed for sensitivity to DNA damage such as UV and gamma radiation, and for interactions with other Haloferax volcanii recombination genes. Novel recombination genes are also being characterised in another archaeal species, Methanothermobacter thermautotrophicus, in collaboration with Dr Ed Bolt.
Haloferax volcanii cells containing the bgaH beta-galactosidase gene stain blue with X-gal
Using these complementary strategies of reverse and conventional genetics, we expect to shed light on novel aspects of recombination in the archaea, and how these organisms meet the physiological challenges posed by the harsh environments they occupy. In addition, Haloferax volcanii may provide a simple test bed for the exploration of eukaryotic systems.