Functional Analysis Of The Homologous Recombination Repair Proteins HerA And NurA In The Thermophilic Archaeon Sulfolobus Islandicus

A number of DNA lesions are generated in each cell every day, among which double-strand breaks ?DSBs? constitute one of the most detrimental types of DNA damage. DSBs could lead to genome instability, cell death, or even tumorigenesis in human, if not repaired timely. Two main pathways are known for DSB repair,homologous recombination repair ?HRR? and non-homologous end joining ?NHEJ?.HRR repairs DSBs using a homologous DNA molecule as a template, resulting in error free DNA repair, whereas NHEJ promotes direct re-ligation of the broken DNA ends in an error-prone manner. In eukaryotes, DSBs generated in the S/G2 phase of the cell cycle are preferentially repaired by HRR pathway, while NHEJ is the favorate pathway to repair DSBs in the G1 phase. Bacteria possess multiple pathways for DSB repair, including RecBCD, the primary HR pathway, SbcC-SbcD, and one backupsystem, RecFOR. In eukaryotes, the HRR pathway is mediated by Mrell-Rad50 proteins, homologs of bacterial SbcD-SbcC. However, numerous proteins and multiple layers of regulation exist to ensure these repair pathways are accurate and restricted to the appropriate cellular contexts, making many important mechanistic details poorly understood in eukaryotes. As the third domain of life, archaea is regarded as a chimaera of bacteria and eukaryotes. Its metabolic pathways and cell structures resemble those of bacteria, whereas the information processing is of the eukaryal type or is more similar to their eukaryal counterparts than those in bacteria.Many archaea live in harsh conditions, such as high temperature, high pressure,extreme pH, or strong radiation, where more DNA damages are expected to be produced to the genomes than in normal environments. However the stability of archaeal genomes is comparable with that in other two domains of life, suggesting that archaea could harbor more efficient DNA repair systems. Study on archaeal DNA repair will provide important clues for that on eukaryotes, because archaeal homologs of Mrell/SbcD-Rad50/SbcC, but not RecBCD or RecFOR, have been identified,indicating the existence of a Mrell-Rad50-mediated HRR pathway in archaea.Eukaryotic Mrell-Rad50 complex exhibits ATPase activity, 3'-5' double-stranded DNA ?dsDNA? exonuclease activity and single-stranded DNA ?ssDNA? endonuclease activity. The MRX/MRN complex ?MR complexed with the third protein Xrs2?Saccharomyces cerevisiae?/Nbsl ?higher eukaryotes?? initially processes broken DNA ends in conjunction with Sae2/CtIP. The subsequent extensive processing is carried out by two parallel pathways, Exol/EXO1 or Dna2/DNA2-Sgsl/BLM,through which a long 3'-tail of ssDNA is generated and the ssDNA is utilized in Rad51-dependent strand exchange in HRR. The activities of archaeal MR complex are similar to that of eukaryotes. The RecQ-like helicase Hjm and the 5'-flap endonuclease which exihibited both endonuclease and 5'-3' exonuclease activities have been identified in archaea; however, it is unclear whether they are involved in dsDNA end resection. Intriguingly, two other genes which encode ATPase/helicase HerA and nuclease NurA, respectively are implicated in HR by their genetic association with mrell and rad50 in thermophilic archaea. This has been supported by biochemical characterization of the encoded proteins that Mre11, Rad50, HerA,and NurA are capable of working in concert to process dsDNA to from a 3'-overhang in vitro. So far, very few genetic studies have been reported, especially for HerA and NurA. In this study, we investigated the functions of HerA and NurA using the well-developed genetic system of the hyperthermophile Sulfolobus islandicus in combination with biochemical characterization, cytologic, and transcriptomics analyses, in order to reveal their in vivo roles and mechanism of these proeins.In the previous study, it has been shown that null mutants were not obtainable for mre11,rad50, herA, and nurA in S. islandicus, suggesting that all of them could be essential for cell viability. In this study their essentiality was further investigated and confirmed by mutant propagation assay. Since neither radA, mre11, rad50, herA, nor nurA mutant in Thermococcus kodakaraensis, another hyperthermophile in Euryarchaea, or radA and hjm in S. islandicus could be isolated, we speculated that HRR may be essential in the thermophilic archaea.To further characterize the essentiality of the archaea-specific proteins, HerA and NurA, mutant genes coding for site-directed mutation at the conserved amino acid residues were constructed and used for genetic complementation in S.islandicus. For HerA, among the six mutants, K154R, D176E, D176N, E356D, E356Q, and R381K,only D176E complemented the deficiency of the wild type HerA, indicating that the Walker A ?K154?, Walker B ?E356? and Arginine finger motif ?R381? as well as the conserved residue D176 of HerA are indispensable for its in vivo functions. For NurA,two central residues related to its nuclease activity, D58 and K202, and two hydrophobic residues involved in the interaction with HerA, 1295 and F300, were chosen for mutagenesis. We showed that none of D58E, D58A, K202R, and K202A was able to complement the wild type NurA, suggesting that the nuclease activity of NurA is essential for cell viability. Furthermore, I295L and F300Y substitutions were found to be able to achieve complementation, whereas 1295E and F300E failed to do so. The interaction between the HerA and NurA mutants I295L, I295E, F300Y, and F300E of S. islandicus were further examined by gel filtration, which revealed that I295L and F300Y maintained the interaction with HerA while I295E and F300E did not. Taken together, these results indicate that both the nuclease activity of NurA and the interaction between NurA and HerA are essential for cell viability.To figure out what activities of mutant proteins that were required for the genetic complementation and to reveal the in vivo roles of the proteins, the wild type and mutant genes of HerA and NurA were cloned and the proteins were expressed in, and purified from E. coli. Biochemical characterization showed that the ATPase activity of HerA?K154R?, D176N, E356Q, and R381K, which failed to exert genetic complementation, were very low or undetectable, suggesting that the ATPase activity is essential for the in vivo functions of HerA. And D176E and E356D contained about 1/7 and 1/5, respectively, of the wild type ATPase activity. However only D176E could complement the deficiency of chromosomal herA, suggesting that the ATPase activity of HerA was not sufficient for its cellular function. Due to failure to detect HerA helicase activity, the DNA degradation activity of the HerA-NurA complex was analyzed. The results showed that the 5'-3' exonuclease activity of E356D-NurA reduced to less than 50% of wild type HerA-NurA while D176E-NurA maintained this activity as high as that of the wild-type. This suggests that efficient 5'-3' exonuclease activity is indispensable for cell viability,which is essential to produce 3'-overhang for HRR and represents the in vivo activity of HerA-NurA in the cell.Further, using protein-specfic antibodies and immunofluorescence microscopy,we examined foci formation of HRR proteins in S. islandicus cells. Under the physiological growth conditions, a majority of cells harbored one or two HerA foci.The number of cells with more than two HerA foci increased after UV-irradiation,suggesting that HerA could be involved in the repair of UV-induced DNA damage.The pattern of NurA foci was similar to that of HerA, while the numbers of RadA foci in most cells were 0 or 1, and did not increase apparently after UV-treatment,indicating that RadA may work differently from HerA-NurA. The results are in agreement with a report on transcription analysis of S. solfataricus P2 which showed that her A operon was up-regulated while radA was not changed upon UV irradiation.To better understand other putative functions of HerA in vivo, this protein was overexpressed in S. islandicus cells. We found that HerA overexpression reduced cell viability and produced abnormal cells with enlarged size and increased DNA contents,as shown by microscopy and flow cytometry. DNA damaging agent assay showed that this strain is as sensitive as the wild type strain to methyl methanesulfonate ?MMS?and cisplatin, while it exhibited higher sensitivity to hydroxyurea ?HU?, an agent revealed to cause G2 arrest in S. islandicus cells, compared with the wild type strain.Microarray analysis showed that genes involved in cell division were down-regulated while the transcription of the genes implicated in chromosome resolution/segregation were also changed in the strain, suggesting that HerA overexpression impair DNA metabolism and resulted in mis-regulation of cell cycle. Recent study has shown that the HerA up-regulation was associated with a novel programmed cell death. HerA seems to be involeved in this novel process in thermophilic archaea.Finally, a S. islandicus strain chromosomally encoding an N-terminal His-tagged Her A was constructed by introducing a his-tag-coding sequence at the 5' end of her A gene. The His-tagged HerA and its putative interaction proteins were purified from S.islandicus cells. NurA as well as two other proteins probably involved in HRR, an ATPase ?SiRe₁432? and a Holliday junction resolvease Hjc ?SiRe 1431?,were identified in the fractions. The interactions between HerA and ATPase and Hjc were confirmed by in vitro pull-down assay. This result provided clues for further investigation into the mechanism of the pathway?s? in which herA proteins are involved.