Characterization Of Two DEXD-box DNA Helicases From The Hyperthermophilic Archaeon Sulfolobus Tokodaii

Many environmental factors and the metabolite of the cell can result in damage to DNA. To prevent the lethal or negative effect brought out by the DNA damage, organisms have developed various pathways to overcome these damages and stabilize the genomes. The genomic DNA of thermophilic archaea living in high temperature should be more likely damaged than that of mesophiles. However, the mutation rates of these organisms are not obviously as high as they should be in such a high temperature. This fact indicates the existence of an effective DNA repair system in thermophilic archaea. What is more, many DNA repair factors found in bacteria and eukaryotes do not exist in archaea. Some DNA metabolism pathways in eukaryotes are still unknown. The DNA metabolism pathways in archaea are relatively simpler than those of eukaryotes, although with more or less similarity. The research work of this field in archaea can provide models for the research in eukaryotes. Since the mutation of DNA repair factors is responsible for several diseases, the research of these proteins may guide the researchers to find ways to treat some diseases.DEXD box is a conserved motif existing in a variaty of DNA and RNA helicases and was named after a Asp-Glu-Ala(X)-Asp core, which is demonstrated to be essential for ATP binding and hydrolysis. RecQ and RecG, two E. coli helicases, along with other helicases, contain this motif. However, in Archaea, no homologue of RecQ and RecG in amino sequences has been found. It has been reported recently that a RecQ functional homologue is discoveried. This fact may be suggestive of the existence of RecG functional homologue in Archaea.In this paper, we cloned, expressed and purified two putative DNA helicases containging DEXD box, ST0590 and ST0147 from Sulfolobus tokodaii Str.7. The preliminary assay showed that these two helicases unwind the double strand DNA with the hydrolysis of ATP. We also observed the promotion effect of unwinding activity of these two helicases by proliferating cell nuclear antigen (PCNA). The interaction between these helicases and PCNA indicates their involvment in DNA replication, albeit with a different promoting efficiency.We further tested the unwinding details of these helicases towards synthesised replication fork DNA and found that they prefer unwinding the nascent strands to the template strand. In this assay, we confirmed that ST0147 unwinds the lagging nascent strand a little more effectively than the leading strand. However, the unwinding preference of ST0590 was not observed.We noticed the failure of these two helicases to completely unwind the double strands DNA when we performed an unwinding assay. This phenomenon may suggest an annealing activity of these helicases. So we tested this hypothesis and confirmed the annealing ability of these proteins and found their differnt annealing efficiency towards several DNA structures.The tendency of these proteins to unwind the nascent strand and anneal the templete strand may suggest a similarity of these helicases to the E.coli RecG, a protein widely believed to promote the replication fork regression. We later demonstrated the similar function of ST0147 in vitro. So we described ST0147 as the functional homologue of RecG in E.coli. We still have to perform further detailed reseach on these two proteins to confirm their functions in vivo.