Abstract
The DNA nuclease gene ST2109 has been cloned from the hyperthermophilic archaeon Sulfolobus tokodaii and expressed in Escherichia coli. The recombinant protein StoNurA has been purified to homogeneity by affinity chromatography and gel filtration chromatography. Biochemical analyses demonstrated that StoNurA exhibited DNA binding and 5'-3' exonuclease activities towards ssDNA and dsDNA. The temperature and pH optima of StoNurA were determined to be 65 °C and 8.0, respectively. The activity of StoNurA was found to be dependent of Mn2+, and its half-life of heat inactivation at 100 °C was 5 min. Gel filtration chromatography revealed that StoNurA could form dimers in solution. Pull-down assays also showed that StoNurA physically interacted with a DNA helicase (StoHerA). Our data suggest that NurA may play a key functional role in the processing of DNA recombinational repair.
Highlights
DNA nucleases can cleave the phosphodiester bonds between the sugar and phosphate moieties of DNA to give a 5’-terminal phosphate and a 3’-terminal hydroxyl group
Correspondence to: Duobin Mao E-mail: duobinmao@126.com. Based on whether they catalyze the cleavage reactions occurring at the ends or within the DNA strands, DNA nucleases can be categorized as exonuclease or endonuclease
We report the cloning, expression and characterization of a Mn2+-dependent DNA nuclease from S. tokodaii (StoNurA) and an evaluation of its role in the archaeal recombinational repair
Summary
DNA nucleases can cleave the phosphodiester bonds between the sugar and phosphate moieties of DNA to give a 5’-terminal phosphate and a 3’-terminal hydroxyl group. A 34-nucleotide ssDNA sequence (5’-CAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGA-3’) labeled with FITC at its 3’ end was used as a substrate for the DNA-binding and nuclease assays, together with the corresponding 34-bp dsDNA. The binding ability of StoNurA to a series of different DNA substrates was examined using a gel shift assay.
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