Biotechnology with a pinch of salt - isolation of DNA repair enzymes from halophilic archaea

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BackgroundHel308 is a monomeric 3' to 5' DNA helicase conserved throughout archaea and metazoans however, is absent from bacteria and fungi. Hel308 is a member of the helicase superfamily 2. An investigation into Drosophila melanogaster mutants which exhibited hypersensitivity to DNA crosslinking agents led to the discovery of Hel308 and consequently the human homologue HELQ. Dependant on the organism of study, Hel308 is known by other names including HELQ in higher eukaryotes, and Hjm in some archaeal species for example Purococcus furiosus and Sulfolobus tokodaii. Though a member of the Ski2 family of helicases, Hel308 acts analogously to RecQ DNA family helicases and has consequently been implicated in a variety of overlapping DNA repair pathways, as well as homologous recombination, and genome stability. Despite these connections, the exact role of Hel308 has remained elusive.OutlineStructural, biochemical, and genetic analyses of Hel308 and its respective gene, have started to unravel the Hel308 mystery. Utilizing both the biochemical and genetic tool-boxes of Haloferax volcanii, protein and genetic interacting partners of Hel308 will be further explored under both native and stress conditions. Additionally, mutations of key residues identified via structural and sequence analysis will be investigated for phenotypic consequences. Overall, the function(s) of and the regulatory mechanism(s) of Hel308 will be examined.Unfortunately, no crystal structure for the Haloferax volcanii Hel308 has been obtained to date, there are however resolved crystal structures of three Hel308 homologues. From these crystal structures Hel308 is shown to be composed of 5 domains around a central DNA binding core which is identifiable by a lining of essential DNA binding residues. The 3' ssDNA overhang threads through this DNA binding core and the five domains drive a 13-hairpin between the two strands subsequently separating the DNA duplex. As with all superfamily 2 helicases Hel308 requires ATP binding and hydrolysis to enable translocation along the DNA. Key residues targeted for mutation include Walker A and B ATPase dead mutations, both independently and in combination with the previously identified hyper-recombinant point mutations.It has recently been shown that in Haloferax volcanii, mutants deleted for radB exhibit a decrease in the level of homologous recombination to approximately 5% of that in wild type cells. However, a double deletion of hel308 with radB restores, in part, homologous recombination. This data suggests an anti-recombinase role for Hel308. Further genetic analysis between hel308 and the homologous recombination pathway will be conducted focussing on the relationship between RadA and Hel308.ImpactThe mammalian Hel308 homologue HELQ, has previously been implicated in genome stability upon the recognition of several certain Hel308 single nucleotide polymorphisms (SNPs) being more prevalent in numerous types of head and neck cancers including esophageal squamous cell carcinoma and gastric adenocarcinoma. In addition to this, it has been suggested that HELQ has a critical role in germ cell maintenance and tumour suppression of ovarian cancers in mammals, indicating a possible role in replication-coupled DNA repair. Due to the evolutionary descent of eukaryotes from archaea, archaea present an opportunity to explore the function of eukaryotic HELQ in a simplified system. By exploring the function of Hel308 in Haloferax volcanii, we can begin to elucidate the mechanism of HELQ and therefore understand better how HELQ affects genome stability and DNA repair. Ultimately this understanding could lead to improved treatments for these cancers.