| DNA double-strand break (DSBs) is one of most severe damages in all organisms. DSBs can be generated either by external agents such as ionizing radiation and mechanical stress or by internal errors during replication and recombination. If not properly processed, DSBs can cause genome instability and cells may develop to cancers in mammalians. Homologous recombination pathway is one of efficient repair processes of DSBs. In bacteria, DSBs are repaired mainly by RecBCD, RecFOR and SbcC-SbcD complexes. In eukarya, DSBs are repaired mainly by Mre11/Rad50-mediated homologues recombination pathway, but the detailed mechansiam of the Mre11/Rad50 complex processing DSB is still obscure. Archaea is the third domain of life. Archaea and bacteria share similar genomic structures and mechanisms of genome duplication, but archaeal repair processes are far more closely related to those in eukarya than to those in bacteria. 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 huaman diseases.In eucarya, the Rad50 and Mre11 proteins in association with a third protein partner (Xrs2 in yeast, Nbs1 in vertebrates) play a key role in the initiation of homologous recombination. Mre11 is a 3'-5' ssDNA (single-stranded DNA) endonuclease and structure specific ssDNA endonuclease. Rad50, an ABC type ATPase, has a long hinge between the amino and carboxyl terminals. Xrs2 and Nbs1 do not share obvious sequence similarities but could be functional analogs. In archaea, which lack both RecBCD and RecFOR homologs, the processing of DSBs may include Rad50 and Mre11 proteins, which are homologs to bacterial SbcC and SbcD proteins, repectively. No homolog of Xrs2/Nbs1 has been found in archaea. Intriguingly, in most archaeal genomes, Mre11 and Rad50 homologs are arranged in an operon-like structure with two recently identified enzymes: a DNA helicase (HerA) and a nuclease (NurA). However, there has been no report so far demonstrating that archaeal NurA and HerA proteins functionally interact with the Mre11-Rad50 complex in the processing of DNA ends in recombination and repair. In this work, the biochemical properties, structure and function of NurA in processing DNA ends were studied in order to understand the mechanism of repair DSBs in recombination and repair.We cloned nurA gene of the hyperthermophilic archaeon Sulolobus tokodaii and expressed the protein (StoNurA) in Escherichia coli. His-tagged StoNurA was purified by heat treatmeat, Ni-NTA affinity and SephacrylTM S-200 HR gel filtration chromatography. StoNurA was found to be hexamers or heptamers based on SDS-PAGE and gel filtration chromatography, unlike momeric and oligomeric structures of reported nucleases. Molecular size of purified StoNurA by SDS-PAGE analysis was 38KDa, which was in agreement with predicted protein size. The enzyme was highly thermostable. It remained active and stable after being treated at 85℃for 30 min. Biochemical analysis demonstrated that StoNurA exhibited both ssDNA endonuclease activity and 5'-3' exonuclease activity on ssDNA and ds DNA (double-stranded DNA). All nucleolytic activities of StoNurA require manganese. The temperature optima of the enzyme was 65℃. The exonuclease activity of StoNurA was not inhibited by low NaCl concentration (0-75mM) and was inhibited by high NaCl concentration (75-300mM). NaCl concentration was not effect the binding activity of StoNurA, even at high concentration (1 M). We examined the DNA binding activity of StoNurA using ssDNA, blunted-ended dsDNA and 3'-overhang as substrates. StoNurA exhibits more strongly binding to 3'-overhang than to ssDNA and blunted-ended dsDNA, which provided evidence that StoNurA might be involved in processing 3'-overhang during the initiation of homologous recombination.In order to study the structure and catalytic residues of StoNurA nuclease, we constructed a series of vectors to express deletion mutants and site-directed mutants of StoNurA. Limited proteolysis experiments were carried out on the purified His-tagged StoNurA protein and a fragment with an estimated size of～30 kDa was obtained by SDS-PAGE analysis. N-terminal sequence of the trypsin-digested fragment was identified by sequential Edman degradation analysis and determined to be Gln-Ile-Ser-Leu-Leu. The sequence corresponded to residues 19-23 of StoNurA for trypsin cleavage. The C-terminal cleavage site was estimated at residues 303 from fragment size and specificity of trypsin for positively charged residues. Based on the N-terminal sequence of trypsin-treated StoNurA and our estimated of the C-terminal cleavage site, we constructed three deletion mutants: StoNurA(19-331), StoNurA(19-303) and StoNurA(1-303).Alignment of StoNurA homologues from archaea revealed three conserved motifs and functions of the mitifs are unclear. Five conserved residues from the three motifs were chosen be changed to alanine for site-directed mutagenesis. Vectors for the mutants D56A, E114A, D131A, Y291A and H299A were constructed. All the mutant proteins were purified as the wild-type StoNurA. We tested the biochemical properties of the mutants. The three deletion mutants StoNurA(19-331), StoNurA(19-303) and StoNurA(1-303) had similar DNA binding and exonuclease activity as wild-type StoNurA. This result demonstrated that removal of N-terminal 19 and C-terminal 28 residues from wild-type StoNurA did not impair catalytic function and StoNurA(19-303) was the core domain structure of StoNurA. The exonuclease activities of site-directed mutants D56A, E114A, D131A and H299A were entirely lost while Y291A was kept only about 30%. The result showed that D56, E114, D131 and H299 may be the catalytic residues of exonuclease activity of StoNurA and Y291 may be involved in DNA binding. Our results may help better elucidate the function of novel nuclease structure-NurA domain.We conducted a Ni-NTA agarose bead pull-down assay to isolate StoNurA-interacting proteins from cell lysates of S. tokodaii. His-tagged StoNurA proteins were incubated with cell lysates of S. tokodaii, and His-tagged StoNurA and its associated proteins were captured by Ni-NTA agarose beads. Four proteins putatively interacting with His-tagged StoNurA were identified by MALDI-TOF mass Spectrometry analysis and determined to be aspartyl-tRNA synthetase, elongation factor 1 (EF1) alpha, sulfidede hydrogenase flavoprotein subunit and Single-Stranded DNA Binding protein (SSB). StoSSB was selected as a candidate of novel StoNurA-interacting protein. In in vitro binding assay, the purified His-tagged StoSSB was incubated with native StoNurA (non-His-tagged) proteins and Ni-NTA agarose beads. In the presence of StoSSB, StoNurA was eluted together with StoSSB. The result strongly supported that StoNurA interacted with StoSSB directly. In order to determine whether StoNurA interacted with StoSSB in vivo, we performed yeast two-hybrid analysis. Interaction between StoNurA and StoSSB was observed when the transformants of both StoNurA and StoSSB genes were assayed on the SD medium without Leu, Trp, and His, and on the SD medium without Leu, Trp, and His, but containing 5 mM 3AT. Even on SD medium without Leu, Trp, His, and Ade, the transformants could also grow. These results demonstrated that StoNurA and StoSSB interacted with each other in vivo. The two-hybrid analysis also showed that StoNurA could form oligomers, which was in agreement with the gel filtration analysis. In co-immunoprecipitation analysis, protein A Sepharose beads conjugated with anti-StoNurA polyclonal antibodies were added to the mixture of StoNurA and StoSSB. StoSSB was found to be co-immunoprecipitated with StoNurA. This result also confirmed that StoNurA physically interacted with StoSSB. We found that StoSSB inhibited the ssDNA and dsDNA exonuclease and the ssDNA endonuclease activities of StoNurA. We assume that this inhibition was mediated through functional interaction between StoNurA and StoSSB. The specificity of the inhibition was tested with SSB from the thermophilic eubacteria Thermotoga maritima (TmaSSB). No significant inhibitory effect on the exonuclease activity of StoNurA towards ssDNA and dsDNA were observed. The result suggests that the inhibitory function of StoSSB is specific for StoNurA. We also found that C-terminal region of StoNurA is important for StoSSB inhibition to the exonuclease activity of StoNurA. Our findings indicate that StoNurA interacts with StoSSB physically and functionally and the two proteins function together in the initiation of homologous recombination. To the best of our knowledge, this is the first report showing interaction of NurA and SSB.In conclusion, in this study, we characterized the biochemical properties of StoNurA and identified the domain structure of StoNurA. We firstly report interaction of NurA and SSB in archaea. Our results would provide evidences to better elucidate the mechanism of Mre11/Rad50-mediated homologues recombination in archaea...
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