硫化叶菌Sulfolobus tokodaii解旋酶HerA的生化性质研究及体内功能的初步分析
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摘要
古菌是与真核生物和细菌并列的第三种生命形式,被认为是真核生物和细菌的嵌合体,其代谢方式类似于细菌而遗传信息的加工则和真核生物类似。古菌大多数生活在高温、高压、极端pH、强离子辐射等极端环境下,其基因组更容易遭受环境的胁迫而产生各种DNA损伤。其中,DNA双链断裂(double-strand breaks,DSBs)是生物体最严重的DNA损伤方式之一,若不修复,将引起生物体基因组不稳定,进而导致生物体死亡。同源重组修复是生物体修复DSBs主要方式之一。在细菌中,同源重组修复主要包括RecBCD、RecFOR和SbcC-SbcD途径。在真核生物中,同源重组修复是由Mre11-Rad50介导的,但具体机制尚不清楚。在古菌中,没有发现RecBCD和RecFOR同源蛋白,但存在SbcC/Mre11和SbcD/Rad50同源蛋白,因此古菌中可能存在Mre11-Rad50介导的同源重组修复。Mre11具有3'-5'ssDNA核酸外切酶和核酸内切酶活性。Rad50是ABC(ATP-binding cassette)型ATPase。在真核生物Mre11-Rad50介导的同源重组修复中,除了Mre11和Rad50外,还有Nbs1(哺乳动物)/Xrs2(酿酒酵母)作为配体,形成MRN/MRX复合体共同参与双链断裂重组修复起始阶段3'-overhang结构的产生过程。而在古菌中,只发现了Mre11和Rad50同源蛋白,没有发现Nbs1/Xrs2配体的同源蛋白。在硫化叶菌Sufolobus tokodaii及其它许多嗜热古菌基因组中,与Mre11和Rad50同一个操纵子内存在另有两个开放阅读框(ORFs)。研究表明,古菌中的这两个ORF其中一个是核酸酶NurA,另一个是解旋酶HerA。HerA是一种新型的解旋酶,广泛存在于嗜热古菌中。其基因在基因组中的组织形式暗示该解旋酶与其关联蛋白Mre11和Rad50之间可能存在着功能上的联系,但是它们之间的关系目前仍然不清楚。NurA、HerA与Mre11/Rad50是否具有相互作用、相互作用的机制以及它们在双链断裂重组修复中的功能还不清楚。本论文旨在研究解旋酶HerA蛋白的体外生化性质、与Mre11之间的相互作用以及在体内可能参与的代谢过程,以期阐明解旋酶HerA在体内参与的生物学过程和功能,从而为真核生物中Mre11/Rad50介导同源重组修复机制提供启示。
本研究中,我们首先克隆了S.tokodaii中HerA基因,并在大肠杆菌中异源表达和纯化了HerA蛋白(StoHerA)。在用分子筛纯化过程中我们发现StoHerA可以以六聚体或者七聚体的形式存在。ATPase活性检测表明StoHerA蛋白在没有DNA存在的情况下也有很高的活性(平均每个HerA分子每分钟水解约4.25个ATP分子)。同时StoHerA的ATPase活性可以被ssDNA,5'-overhang和钝端双链DNA(blunt-ended DNA)激活,并且更依赖双链DNA,与S.acidocaldarius和Pyrococcus abyssi中的同源蛋白类似:DNA结合实验表明,StoHerA更倾向结合具有次级结构的Holliday junction(HJ)和splayed-arm DNA(Y-型DNA);解旋酶活性检测发现StoHerA能够高效地解开HJ和splayed-arm DNA底物,也能对钝端双链进行解链,但是解链效率较低。
同时,我们克隆了S.tokodaii中Mre11基因,在大肠杆菌中异源表达并纯化了StoMre11蛋白,通过pull-down和酵母双杂交的方法,证明了StoHerA和StoMre11之间存在着相互作用,并且HerA的ATPase活性和解旋酶活性可以被Mre11所激活。
另外,我们对HerA的四个保守位点进行了点突变分析,发现位于Walker A和Walker B保守结构域的K153或E355突变为丙氨酸会导致其ATP酶活性和解旋酶活性丧失,而D175A和R380A定点突变体的ATP酶活性和解旋酶活性则大大减弱。其中,有意思的是位于walker B保守结构域的E355位点,该位点突变为丙氨酸,不仅导致ATP酶活性和解旋酶活性的丧失,而且也导致DNA结合能力的丧失。
其次,我们利用目前应用较广的两种穿梭载体-基于病毒载体SSV1构建的病毒载体pMJ0503和基于质粒pRN1构建的质粒载体pJ-在古菌S.solfataricusPH1-16中分别表达带有组氨酸标签的StoHerA及其点突变体StoHerA(D175A)蛋白,并利用Ni~(2+)-NTA琼脂糖柱进行纯化,免疫印迹检测及质谱鉴定结果表明野生型StoHerA及其突变体StoHerA(D175A)蛋白在S.solfataricus PH1-16中得到了表达;同时在用Ni~(2+)-NTA琼脂糖柱纯化StoHerA的洗脱液中我们也发现了一些可能与StoHerA结合的蛋白,对SDS-PAGE的蛋白条带进行质谱鉴定,结果表明其中一些蛋白与DNA代谢相关,如核酸酶NurA(SSO2248)、古菌REP家族蛋白(SSO1515)等:另外一些蛋白与糖脂代谢相关,如假想的酯酶(SSO2979),酰基辅酶A合成酶(SSO1806),葡萄糖脱氢酶(SSO3204)和赖氨酰tRNA合成酶(SSO2979)等。对于病毒载体我们也在洗脱液中发现了一些可能结合的蛋白,包括DNA代谢相关蛋白Rep(SSO1515);信号转导蛋白(SSO0029);酰基辅酶A代谢相关蛋白酰基辅酶A合成酶(SSO1806)和酰基辅酶A脱氢酶(SSO2877);糖脂代谢相关蛋白古菌果糖1,6-二磷酸酶(SSO0286),假想的酯酶(SSO2979)以及氨基酸合成相关的赖氨酰tRNA合成酶(SSO2979)等。以上对SDS-PAGE蛋白条带的质谱鉴定结果暗示着StoHerA可能在体内和这些蛋白之间存在着直接或间接的相互作用。
接着我们通过二维双向电泳(2-DE)的方法对洗脱液中的成分进行进一步分离。虽然由于样品选择的原因,洗脱液中的样品通过2-DE分析后我们没有发现HerA蛋白,但是一些DNA损伤相关蛋白如单链结合蛋白SSB(SSO2364)、DNA解旋酶XRCC2/XPD(Ta0057)、ASNC家族RNA聚合酶、转录因子(SSO0606)和TenA家族转录激活子(SSO2089)出现在质粒载体表达系统的样品中。而在病毒载体表达系统中我们发现了ABC转运系统ATP结合蛋白SS03093,这个结果暗示了StoHerA可能与该系统蛋白相互作用,同时也暗示着StoHerA可能参与了一些物质的转运过程,这个推测需要进一步的证据来证实。
免疫共沉淀的结果显示在HerA过量表达的S.solfataricus PH 1-16体内StoHerA和酰基辅酶A合成酶(SSO1806)一块被其兔血清抗体免疫共沉淀下来,说明这两个蛋白可能存在直接或间接的相互作用,这与S.solfataricus PH 1-16中StoHerA蛋白的表达时鉴定的结果一致。
遗传分析表明,在S.solfataricus PH 1-16表达HerA蛋白的转化子中,阿拉伯糖基因和HerA基因ST2106一块整合到S.solfataricus PH 1-16的基因组上。而基因组中插入突变的pyrF基因则可能发生了回复突变或者是与载体上的野生型pyrF基因发生了同源交换。
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 motifⅢmight 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 Ni~(2+)-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.
本研究中,我们首先克隆了S.tokodaii中HerA基因,并在大肠杆菌中异源表达和纯化了HerA蛋白(StoHerA)。在用分子筛纯化过程中我们发现StoHerA可以以六聚体或者七聚体的形式存在。ATPase活性检测表明StoHerA蛋白在没有DNA存在的情况下也有很高的活性(平均每个HerA分子每分钟水解约4.25个ATP分子)。同时StoHerA的ATPase活性可以被ssDNA,5'-overhang和钝端双链DNA(blunt-ended DNA)激活,并且更依赖双链DNA,与S.acidocaldarius和Pyrococcus abyssi中的同源蛋白类似:DNA结合实验表明,StoHerA更倾向结合具有次级结构的Holliday junction(HJ)和splayed-arm DNA(Y-型DNA);解旋酶活性检测发现StoHerA能够高效地解开HJ和splayed-arm DNA底物,也能对钝端双链进行解链,但是解链效率较低。
同时,我们克隆了S.tokodaii中Mre11基因,在大肠杆菌中异源表达并纯化了StoMre11蛋白,通过pull-down和酵母双杂交的方法,证明了StoHerA和StoMre11之间存在着相互作用,并且HerA的ATPase活性和解旋酶活性可以被Mre11所激活。
另外,我们对HerA的四个保守位点进行了点突变分析,发现位于Walker A和Walker B保守结构域的K153或E355突变为丙氨酸会导致其ATP酶活性和解旋酶活性丧失,而D175A和R380A定点突变体的ATP酶活性和解旋酶活性则大大减弱。其中,有意思的是位于walker B保守结构域的E355位点,该位点突变为丙氨酸,不仅导致ATP酶活性和解旋酶活性的丧失,而且也导致DNA结合能力的丧失。
其次,我们利用目前应用较广的两种穿梭载体-基于病毒载体SSV1构建的病毒载体pMJ0503和基于质粒pRN1构建的质粒载体pJ-在古菌S.solfataricusPH1-16中分别表达带有组氨酸标签的StoHerA及其点突变体StoHerA(D175A)蛋白,并利用Ni~(2+)-NTA琼脂糖柱进行纯化,免疫印迹检测及质谱鉴定结果表明野生型StoHerA及其突变体StoHerA(D175A)蛋白在S.solfataricus PH1-16中得到了表达;同时在用Ni~(2+)-NTA琼脂糖柱纯化StoHerA的洗脱液中我们也发现了一些可能与StoHerA结合的蛋白,对SDS-PAGE的蛋白条带进行质谱鉴定,结果表明其中一些蛋白与DNA代谢相关,如核酸酶NurA(SSO2248)、古菌REP家族蛋白(SSO1515)等:另外一些蛋白与糖脂代谢相关,如假想的酯酶(SSO2979),酰基辅酶A合成酶(SSO1806),葡萄糖脱氢酶(SSO3204)和赖氨酰tRNA合成酶(SSO2979)等。对于病毒载体我们也在洗脱液中发现了一些可能结合的蛋白,包括DNA代谢相关蛋白Rep(SSO1515);信号转导蛋白(SSO0029);酰基辅酶A代谢相关蛋白酰基辅酶A合成酶(SSO1806)和酰基辅酶A脱氢酶(SSO2877);糖脂代谢相关蛋白古菌果糖1,6-二磷酸酶(SSO0286),假想的酯酶(SSO2979)以及氨基酸合成相关的赖氨酰tRNA合成酶(SSO2979)等。以上对SDS-PAGE蛋白条带的质谱鉴定结果暗示着StoHerA可能在体内和这些蛋白之间存在着直接或间接的相互作用。
接着我们通过二维双向电泳(2-DE)的方法对洗脱液中的成分进行进一步分离。虽然由于样品选择的原因,洗脱液中的样品通过2-DE分析后我们没有发现HerA蛋白,但是一些DNA损伤相关蛋白如单链结合蛋白SSB(SSO2364)、DNA解旋酶XRCC2/XPD(Ta0057)、ASNC家族RNA聚合酶、转录因子(SSO0606)和TenA家族转录激活子(SSO2089)出现在质粒载体表达系统的样品中。而在病毒载体表达系统中我们发现了ABC转运系统ATP结合蛋白SS03093,这个结果暗示了StoHerA可能与该系统蛋白相互作用,同时也暗示着StoHerA可能参与了一些物质的转运过程,这个推测需要进一步的证据来证实。
免疫共沉淀的结果显示在HerA过量表达的S.solfataricus PH 1-16体内StoHerA和酰基辅酶A合成酶(SSO1806)一块被其兔血清抗体免疫共沉淀下来,说明这两个蛋白可能存在直接或间接的相互作用,这与S.solfataricus PH 1-16中StoHerA蛋白的表达时鉴定的结果一致。
遗传分析表明,在S.solfataricus PH 1-16表达HerA蛋白的转化子中,阿拉伯糖基因和HerA基因ST2106一块整合到S.solfataricus PH 1-16的基因组上。而基因组中插入突变的pyrF基因则可能发生了回复突变或者是与载体上的野生型pyrF基因发生了同源交换。
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 motifⅢmight 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 Ni~(2+)-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.
引文
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