Biochemical Characterization And In Vivo Function Of The Novel Helicase HerA From The Hyperthermophilic Archaeon Sulfolobus Tokodaii

Archaea,like bacteria and eukaryotes,is a fundamentally distinct domain of life and it has been proved to be a mosaic of bacteria and eukaryotes:their core metabolic pathways resemble those of bacteria whereas their information-processing processes are distinctly eukaryotic.These organisms commonly reside in harsh conditions,like extremely high temperature,pressure,low pH or strong ionizing irradiation,or a combination of these,that threaten genome stability and produce various DNA damages.DNA double-strand break(DSB) is one of most severe damages in all organisms.If not properly processed,DSBs can cause genome instability and cells may turn to death.Organisms have evolved conserved mechanisms to repair DSBs, homologous recombination is one of the efficient pathways for processing DSBs.In bacteria,DSBs are repaired mainly by RecBCD,RecFOR and SbcC-SbcD complexes. Whereas eukaryotic Mre11/Rad50 complex(MR) with a third partner,Xrs2 in yeast and Nbs1 in vertebrates,is one of the main complexes that are involved in this process. However,the detailed mechanism is obscure.Mre11 and Rad50 homologues are present and conserved in the genomes of thermophilic archaea.Intriguingly,in many genomes of thermophilic archaea,Mre11 and Rad50 homologues are arranged in tandem together with two genes encoding a DNA helicase(HerA) and a nuclease (NurA).This gene organization indicates that HerA may functionally interact with this conserved complex.However,how HerA and NurA interact with Mre11 and Rad50 and what roles HerA and NurA play in Mre11/Rad50-mediated recombination pathway are still unclear now.In the present study,we will focus on the characterization of HerA from the hyperthermophilic archaeon Sulfolobus tokodaii (StoHerA) and the interaction between StoHerA and its related protein StoMre11 (Mre11 from S.tokodaii).By over-expression of StoHerA(or D175A) in the S. solfataricus PH1-16,we try to elucidate the function of StoHerA and biological pathway it may be involved in in vivo.These studies will give clues for the understanding of MRX complex-initiated DSB repair in eukarya.Firstly,we cloned herA gene from the hyperthermophilic archaeon S.tokodaii and expressed the protein(StoHerA) in Escherichia coli.After heat treatment and loading onto a Hitrap Q column,StoHerA was further purified by a superdex 200 gel filtration chromatography.Gel filtration analysis showed that StoHerA might form a hexamer or heptamer in solution.In order to know more about the biochemical properties of StoHerA,we assayed StoHerA enzymatic activities.Strong ATP hydrolysis activity was detected in the absence of DNA(4.25 ATP min^-1 was hydrolyzed by one StoHerA molecule on the average).While 5'-overhang and blunt-ended dsDNA enhanced the ATPase activity strongly,only a slight stimulation was detected by ssDNA.These results showed that StoHerA was similar to HerA from S.acidocaldarius and MlaA(a homologue of HerA from Pyrococcus abyssi),regarding to the DNA dependence of the ATPase activity.The DNA binding ability of StoHerA was then determined by gel shift assay.The results indicated that StoHerA,like MlaA,prefers DNA substrates that have secondary structures.The helicase activity was assayed using dsDNA with 3'-overhang,5'-overhang,blunt-ended DNA,Holliday junction,and splayed-arm DNA substrates.StoHerA was able to unwind all of the substrates tested.It is interesting that StoHerA unwounds blunt-ended dsDNA,although with relatively low ability.Because in Mre11/Rad50 mediated repair of DSBs,a helicase should be present to generate 3'-overhang.StoHerA is therefore suitable to fulfil this function together with NurA,a 5'-3' nuclease linked to Mre11/Rad50.This result may indicate that the enzyme unwinds duplex DNA by direct interactions(helix destabilizing or active mechanism).Besides,StoHerA was able to unwind Holliday junction and splayed-arm DNA efficiently.These results indicate that StoHerA may be involved in the processing of recombination intermediates,which occurs in late stage of dsDNA break repair.StoHerA may also function in the migration of Holliday junctions,a process requiring enzymes binding and unwinding two dsDNA arms.Meanwhile,the mre11 gene was cloned from the hyperthermophilic archaeon S. tokodaii and expressed in E.coli.By pull-down assay and yeast two-hybrid analysis, we showed that the StoMre11 and StoHerA proteins had physical interaction.In addition,the ATPase and helicase activity of StoHerA were stimulated by StoMre11. In mammalian cells,it has been proposed that Mre11 is recruited to DNA damage site for repair,and possibly functions as part of the damage-signalling apparatus.It might be possible that,in archaea,through interaction with StoHerA,StoMre11 helps StoHerA localize to break point and improves its dsDNA unwinding activity.Furthermore,by site-directed mutagenesis,we found that the residues in the conserved motifs were all related to ATPase and helicase activities.Among them, K153 and E355 were essential for ATP hydrolysis.Interestingly,the conserved glutamic acid in Motif B was not only critical for the ATPase activity,but also essential for dsDNA binding.While loss or reduction of the ATPase or helicase activity of K153A,D175A,and R380A were perhaps solely related to the ATP hydrolysis,loss of the ATPase or helicase activity of E355A might be due to either the inactivation of ATP hydrolysis or the inability of dsDNA binding,or both.These results suggested that E355 and motifâ…¢might be involved in coordination among ion chelating,ATP binding and hydrolysis,dsDNA binding,and helicase activities.Secondly,we used the modified plasmid vector pJ to express StoHerA in S. solfataricus PH1-16.The transformants were obtained and StoHerA protein was purified by Ni²⁺-NTA agarose column chromatograph.Interestingly,a few closely related proteins were eluted together with StoHerA,for example,SsoNurA (SSO2248),Archaeal PaREP1,PaREP8(SSO1515).And some seemingly unrelated proteins,for example,esterase_lipase(SSO2979),fructose-1,6-bisphosphatase (SSO0286),were also eluted.Using the same method,the viral vector pMJ0503-StoHerA(D175A) was transformed into S.solfataricus PH1-16 for expression his-tagged D175A.A few proteins were co-eluted with D175A in the elution.These proteins included Archaeal PaREP1,PaREP8(SSO1515), signal-transduction protein(SSO0029),acyl-CoA synthetase(SSO1806),acyl-CoA dehydrogenase(SSO2877),esterase lipase(SSO2979),fructose-1,6-bisphosphatase (SSO0286) and lysyl-tRNA synthetase(SSO0090).Further,we performed two-dimension electrophoresis(2-DE) to analysis these proteins which were co-eluted with StoHerA.We did not find the StoHerA protein in sample from either plamid vector or viral vector.However,proteins such as single-stranded DNA binding protein(SSB)(SSO2364),AAA⁺ ATPase family protein (SSO0176),RNA polymerase transcription factors(SSO0606) and TenA family transcription regulator(SSO0606),which might be involved in DNA repair,were found in the sample from the plasmid vector.In the sample uisng the viral vector system,we found ABC transporter ATP binding protein(SSO3093) and AAA ATPase family protein(SSO0176) besides some seeming unrelated protein.Moreover,by co-immunoprecipitation assay,we found that acyl-CoA synthetase (SSO1806) was able to be co-immunoprecipitated with StoHerA or SsoHerA.This result suggested that acyl-CoA synthetase(SSO1806) might interact with StoHerA or SsoHerA in vivo,in accordance with the results from 2-DE.By PCR with designed primers,we found that the arabinose promoter together with the gene for StoHerA has been integrated into the genome,while the wild type gene for pyrF in the vector has replaced the pyrF in the S.solfataricus PH1-16 which was mutated by a insertion sequence.This results indicated that our vector based on pJ might potentially be used for gene knock out which could be suitable for uracil selection,thus had a wild host range for application,although we need to investigate the location in the future.