Characterization Of The Structure And Function Of A Novel ATPase In Sulfolobus Islandicus

Archaea is the third domain of life.The cellular structure and metabolic pathways of archaea resemble those of bacteria whereas the genetic information processing and transmission mechanisms,including those for DNA replication,transcription,recombination,repair and protein translation,are more closely releated to those of eukarya.Sulfolobus,one of the model of thermophilic archaea,thrive in high temperature and acidic environment.However,their genetic integrity is well maintained and the spontaneous mutation rate is not higher than that of mesophile.The obvious paradox implies that Sulfolobus cells contain an efficient DNA repair system.Until now,three DNA repair pathways have been identified in Sulfolobus cells:homologous recombination repair,nucleotide excision repair,and base excision repair.In addition,the archaeal proteins which take participate in DNA metabolism are more similar to the eukaryotic homologs than those of bacteria.DNA double-strand break?DSB?is one of the most serious DNA damages in vivo.Two important pathways are utilized for DSBs damage repair:homologous recombination repair and non-homologous end joining.So far,homologous recombination repair has been found in archaea and a series of elegant studies on Mre11,Rad50 and RadA have been reported.Besides these conserved proteins,there are also reports on archaea specific homologous recombination repair proteins,such as NurA,HerA,Hjc,and Hje.Holliday junction is a hallmark intermediate in DNA recombination,which is required to be processed for proper DNA repair and chromosome segregation.HJ can be processed in two different ways.One is resolution,which is mediated by HJ resolvase.HJ resolvases have been identified in all three domains of life,including RuvC in bacteria,Hjc in archaea,and Yenl/GEN1,and Slxl-Slx4/SLX1-SLX4 in eukarya.Another pathway is called dissolution,which involves HJ migration enzyme.In E.coli,RuvAB complex is responsible for HJ recognition and migration.However,the equivalents of the E.coli Ruv AB migration motor have not been reported in eukarya or in archaeaIn this study,we expressed a C-terminal His-tagged Hjc in S.islandicus.Through affinity purification of His-tagged Hjc and mass spectrum analysis of the co-purified proteins,we identified a protein encoded by SiRe₁432.The gene is located at the same locus with that encoding Hjc?SiRe₁431?in the S.islandicus REY15A genome.Bioinformatic analysis of the SiRe₁432 revealed that the protein is composed of a classical PIN domain and a P-loop ATPase domain.Therefore,we named this protein as SisPINA,for PIN domain-containing P-loop ATPase from Sulfolobus islandicus.We confirmed the physical interaction between SisPINA and Hjc by pull-down analysis.The interaction between Hjc and PINA implies that SisPINA and Hjc are associated with processing HJ in vivo.To explore the function of SisPINA in S.islandicus cells,we attempted to knock out the chromosomal gene encoding SisPINA.After several rounds of counter-selection,we did not obtain any colony of cells with the SisPINA gene deleted,implying that the cell cannot survive without SisPINA.In addition,we performed genetic complementation experiment and confirmed that SisPINA is an essentional gene for S.islandicus cells survive.Subsequently,we characterized the biochemical activities of SisPINA.First,we cloned SisPINA gene and and constructed plasmids to express the wild type and four site-directed mutants.We expressed SisPINA and its mutant proteins in coli and purified the proteins.Using the purified SisPINA,we performed ATPase,DNA binding,and branch migration assays.The results clearly showed that SisPINA can hydrolyze ATP.However,the ATPase activity of SisPINA was not stimulated by various DNA substrates,including ssDNA,dsDNA,5'-overhang DNA,3'-overhang DNA,Y-DNA,replication fork DNA,and Holliday junction DNA.Intriguingly,the results by EMSA?Electrophoretic Mobility Shift Assay?indicate that SisPINA can bind all the DNA substrates but only forms stable protein-DNA complexes with replication fork and HJ DNA.In addition,we confirmed that SisPINA is able to promote branch migration and unwind Y-shaped DNA powered by ATP hydrolysis.To further study the role of SisPINA in vivo,we identified the proteins which interact with SisPINA and analysed the significance of these interaction.We found that SisPINA can stimulate the cleavage activity of Hjc on a fixed HJ substrate and SisPINA can also enhance the cleavage of the preferred strand by Hjc during mobile HJ resolution.However,HJ branch migration by SisPINA is inhibited by Hjc.Besides the interaction with Hjc,SisPINA can also form stable complex with RFCs and Hjm,respectively.Furthermore,the pull-down assay revealed that the C-terminal region of SisPINA plays a key role for these interaction.In order to further characterize SisPINA at the molecular level,we determined the crystal structures of two SisPINA mutants after many trials failed to obtain high-quality crystals of the wild-type SisPINA protein.The per asymmetric unit cell of SisPINAK261A crystal contained six SisPINAK261A molecules.The six SisPINA molecules assemble into a hexameric ring,which is in agreement with the results of size-exclusion chromatography.Further analysis of the structures of the six subunits revealed that two different conformations exited.Subunit A?and subunit B,C,and E with the same conformation?represents the SisPINA conformation without ATP while subunit D?and F?represents the ATP-binding conformation.Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration.Due to lack of the electron density,we missed the C-terminal region in the SisPINAK261 A structure.In the structure of SisPINAR147KI199SR206A,the C-terminal of SisPINA was folded into a KH domain(?hnRNP K homology domain?.Intriguingly,the unit cell of SisPINAR147KI199SR206A contained only one molecule,the mechanism of this is still elusive.Based on the structure of SisPINA and Hjc,and the functional interaction between SisPINA and Hjc,we proposed a model to explain how SisPINA and Hjc process HJ DNA together.This model provides new sight into the mechanism of HJ processing in vivo.