【作者】 魏涛；

【导师】 申玉龙；

【作者基本信息】 山东大学 ， 微生物学， 2008， 博士

【摘要】 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和人类中的癌症发生,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Mre11-Rad50介导的修复途径,但具体机制尚不清楚。古菌作为生命的第三种形式,在基因组结构方面与细菌相似,而DNA修复机制与真核生物更相似。研究古菌DNA修复机制,有助于揭示真核生物DNA修复分子机制,进而为多种人类疾病的预防与治疗提供相应的理论指导。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核酸外切酶和底物结构特异性ssDNA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bite11和Rad50外,还有Nbs1（哺乳动物细胞）/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结构产生的过程。而在古菌中,只发现了Mre11和Rad50蛋白,没有发现Nbs1/Xrs2配体。在Sulfolobus tokodaii及其它古菌基因组中,与Mre11和Rad50同一个操纵子内另有两个开放阅读框（ORFs）。研究表明,古菌中的这两个ORF其中之一是新型核酸酶NurA,另一个解旋酶HerA。到目前为止,NurA、HerA与Mre11和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的功能还不清楚。本论文旨在研究NurA生化性质、蛋白结构以及在重组修复中具体功能,进而揭示古菌双链DNA断裂重组修复机制。本文克隆了超嗜热古菌S.tokodaii的nurA基因并在大肠杆菌中表达了NurA蛋白（StoNurA,331 a.a.）,经热处理,Ni-NTA柱亲和层析和分子筛层析纯化,得到纯化的重组StoNurA蛋白。分子筛(sephacryl^TMS-200 HR)纯化过程中,发现StoNurA蛋白形成六聚体或七聚体大分子形式,与已报道的核酸酶以单体和二聚体存在不同。SDS-PAGE结果显示,StoNurA蛋白单体分子量为38.0 KDa,与预期的大小一致。纯化的StoNurA蛋白在85℃处理30 min,仍然具有很好的热稳定性。生化活性分析表明,StoNurA蛋白具有5’-3’ssDNA（single-stranded DNA）和dsDNA（double-stranded DNA）核酸外切酶及单链内切酶活性。该酶是Mn²⁺离子依赖型核酸酶,最适反应温度为65℃。NaCl浓度对StoNurA蛋白核酸酶活性影响较大,NaCl浓度为0-75 mM对StoNurA蛋白外切酶活性没有影响,NaCl浓度为75-300 mM抑制外切酶活性;Natl浓度（0-1.0 M）对StoNurA蛋白DNA结合活性没有影响。StoNurA蛋白可以结合单链、钝端双链和3’-overhang双链底物,其中与与3’-overhang双链结合能力最强,为该酶可能参与3’-overhang产生过程提供间接证据。为了研究StoNurA蛋白结构域构造和催化位点,构建了一系列的缺失突变体和点突变体。我们采用胰蛋白酶限制性水解StoNurA蛋白,得到分子量约为30.0KDa稳定的蛋白片段,该水解片段具有很好的稳定性。通过蛋白质N端测序,发现该水解片段N端为Gln-Ile-Ser-Leu-Leu,位于StoNurA蛋白的第19-23位氨基酸。根据该水解片段分子量大小和胰蛋白酶酶切位点（赖氨酸或精氨酸残基的羧基参与形成的肽键）,确定该水解片段c端位于StoNurA蛋白303位氨基酸。根据水解片段在StoNurA蛋白氨基酸位置,我们构建三个缺失突变体StoNurA（19-331）,StoNurA（19-303）和StoNurA（1-303）。通过利用ClustalW程序进行同源序列比对,发现古菌NurA同源蛋白中存在3个保守motifs,从3个motifs中选取保守氨基酸位点,突变为丙氨酸,构建了表达五个StoNurh定点突变体蛋白D56A、E114A、D131A、Y291A和H299A的载体。将载体转入大肠杆菌中表达蛋白,经热处理,Ni-NTA柱亲和层析和分子筛层析纯化,得到纯化突变体蛋白。对缺失突变体和点突变体蛋白进行了生化活性分析。三个缺失突变体NurA（19-331）,StoNurA（19-303）和StoNurA（1-303）都具有DNA结合活性和核酸外切酶活性。StoNurA（19-303）具有较高的活性,表明去除StoNurA蛋白N端19个氨基酸和C端28个氨基酸,不会破坏StoNurA蛋白催化功能,该突变体是StoNurA蛋白核心结构域。点突变体D56A、E114A、D131A和H299A完全丧失外切酶活性,Y291A保持30%活性,这个结果表明D56、E114、D131和H299可能是StoNurA核酸酶的催化位点,Y291可能与DNA底物结合有关。缺失突变体和点突变体生化分析结果可以很好地解释新型核酸酶结构域NurA domain的生化功能。采用Ni-NTA琼脂糖珠pull-down方法,将带组氨酸标签的StoNurA蛋白与裂解的S.tokodeii菌体全蛋白混合,用Ni-NTA琼脂糖珠捕捉StoNurA形成的蛋白复合物,发现四个可能与StoNurA相互作用的蛋白。通过质谱鉴定分析,这四个蛋白分别为天冬酰胺tRNA合成酶、硫化物黄素蛋白脱氢酶亚基、翻译延伸因子EF1α和单链结合蛋白（Single-Stranded DNA Binding protein,SSB）。根据这四种蛋白质功能,单链结合蛋白StoSSB被推定为与StoNurA相互作用的蛋白。在蛋白体外结合实验中,纯化的StoNurA（不带组氨酸标签）可以通过带组氨酸标签的StoSSB蛋白与Ni-NTA琼脂糖珠结合,证实了StoNurA与StoSSB体外相互作用。我们采用酵母双杂交和免疫共沉淀方法,研究StoNurA与StoSSB生物体内相互作用。在酵母双杂交试验中,阳性克隆（转化pGADT7--StoNurA/pGBKT7-StoSSB和pGADT7-StoSSB/pGBKT7-StoNurA）可以在三种缺陷型SD-His/Leu/Trp,SD-His/Leu/Trp+5 mM 3-AT和SD-Ade/His/Leu/Trp的平板上生长,这个结果显示StoNurA与StoSSB之间生物体内较强相互作用。酵母双杂交结果也证实StoNurA蛋白自身可以相互作用,形成同源聚体,与分子筛结果一致。在免疫共沉淀实验中,StoSSB蛋白可以被StoNurA抗原抗体复合物共沉淀下来,证实StoNurA与StoSSB之间生物体内存在直接的相互作用。生化性质分析结果表明,StoSSB抑制StoNurA的ssDNA和dsDNA核酸外切酶和ssDNA内切酶活性。以嗜热细菌Thermotoga maritima单链结合蛋白TmaSSB作为对照,发现TmaSSB对StoNurA核酸外切酶活性没有抑制作用,这个结果表明StoSSB对StoNurA活性抑制作用具有种属特异性。同时我们还鉴定出StoNurA与StoSSB相互作用结构域位于StoNurA蛋白的C端。这些结果证实StoNurA与StoSSB之间存在物理的和功能的相互作用,为StoNurA与StoSSB共同参与同源重组修复起始阶段提供了证据。这是在古菌中首次发现StoNurA与StoSSB之间相互作用。本论文报道了超嗜热古菌S.tokodaii新型核酸酶StoNurA生化性质、结构域构造和与单链结合蛋白StoSSB相互作用的研究,对于揭示古菌中Mrell和Rad50介导的双链DNA断裂重组修复具有重要意义。更多还原

【Abstract】 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 Sephacryl^TM 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更多还原