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1.
极端嗜热古菌———芝田硫化叶菌 DNA 连接酶 (Ssh 连接酶 ) 的最适辅因子为 ATP ,在 dATP 存在时,该酶也能表现出较弱的连接活性 . ATP 或 dATP 都能够使该酶发生腺苷化,腺苷化的 Ssh 连接酶能够将腺苷基团转移至含切刻的 DNA 上 . 电泳迁移率改变实验表明, Ssh 连接酶能够结合双链 DNA ,且与含切刻及不含切刻的 DNA 结合的亲和力相同,但不结合单链 DNA. 酵母双杂交实验显示,硫磺矿硫化叶菌 ( 与芝田硫化叶菌亲缘关系很近 ) 的 DNA 连接酶,与该菌所含的 3 个增殖细胞核抗原 (PCNA) 同源蛋白中的一个 (PCNA-1) 有相互作用,而与另外 2 个同源蛋白 (PCNA-like 和 PCNA-2) 则无相互作用 . 在古菌中高度保守的 Sac10b 蛋白家族成员 Ssh10b 能够激活 Ssh 连接酶的活性,而硫化叶菌中的主要染色体蛋白——— 7 ku DNA 结合蛋白 (Ssh7) 则对该酶活性没有影响 . 相似文献
2.
反向旋转酶是一种I型拓扑异构酶,它可以利用ATP水解的能量向DNA分子中引入正超螺旋。通过阴离子交换层析、亲和层析、聚丙烯酰胺凝胶电泳(SDSPAGE)从芝田硫化叶菌(Sulfolobus shibatae)中分离得到一种反向旋转酶。SDSPAGE 显示,该酶分子量约为126 kD,N末端序列测定结果表明,该酶为芝田硫化叶菌中一种新的反向旋转酶。 相似文献
3.
通过SPSepharose,DNA纤维素和磷酸纤维素等柱层析 ,从极端嗜热古菌———芝田硫化叶菌 (Sulfolobusshibatae)中纯化得到分子量为 11.5ku的DNA结合蛋白Ssh12 .Ssh12约占细胞总蛋白的 4% .该蛋白既能与负超螺旋DNA也能与松弛DNA结合 .利用含单切刻环状DNA进行的切刻闭合分析表明 ,Ssh12在与DNA结合时能够固定负超螺旋 .这种能力在室温 ( 2 2℃ )下很弱 ,而在 3 7℃以上则大大增强 .Ssh12的细胞内含量和固定负超螺旋的能力提示 ,该蛋白对于芝田硫化叶菌染色体DNA的组织以及热稳定性起着重要作用 . 相似文献
4.
极端嗜热古菌———芝田硫化叶菌(Sulfolobus shibatae)基因组含一对亲缘关系较远的同源基因,ssh10b和ssh10b2。这对同源基因编码的蛋白(Ssh10b和Ssh10b2)属于古菌Sac10b DNA结合蛋白家族。关于Ssh10b以及与其极为相似的硫矿硫化叶菌(S.solfataricus)Sso10b、嗜酸热硫化叶菌(S.acidocaldarius)Sac10b蛋白已有较多研究,推测这些蛋白可能在染色体组织和包装、DNA重组、基因表达调控等方面起作用。克隆并在大肠杆菌中表达了ssh10b2基因,纯化了重组Ssh10b2蛋白。免疫印迹定量分析表明,ssh10b2在芝田硫化叶菌中有表达,但其细胞含量仅相当于Ssh10b的约十分之一。重组Ssh10b2对双链DNA的亲和力低于Ssh10b。此外,Ssh10b2和Ssh10b在与双链DNA结合时表现出相似的凝胶阻滞模式。有意思的是,Ssh10b2固定DNA负超螺旋的能力明显低于Ssh10b。这些结果提示,Ssh10b和Ssh10b2可能具有不同的生理作用。 相似文献
5.
嗜酸热古菌——芝田硫化叶菌(Sulfolobus shibatae)合成大量7 kD DNA结合蛋白Ssh7. Southern杂交结果显示, 该菌基因组中含有两个编码Ssh7蛋白的基因, 分别命名为ssh7a和ssh7b. 对这两个基因进行了克隆、序列测定和在大肠杆菌中的表达. 测序结果表明, 两个Ssh7多肽仅在3个氨基酸位置上有差异; 此外, ssh7a和ssh7b的顺式调控序列也十分相似, 提示存在着维持两个基因的序列和表达都不变的选择压力. 杂交结果还显示, 与芝田硫化叶菌一样, 硫磺矿硫化叶菌(Sulfolobus solfataricus)基因组中也含有两个编码7 kD蛋白的基因. 结合其他报道, 这一结果提示编码7 kD蛋白的基因可能在硫化叶菌种间分歧形成之前发生了基因复制. 采用电泳迁移率改变试验(EMSA)分析了天然及重组Ssh7蛋白与双链DNA片段之间的相互作用. 天然和重组蛋白的EMSA行为相似, 说明Ssh7与DNA的相互作用既不受发生在天然蛋白赖氨酸残基上的甲基化的影响, 也不受两种多肽异构体之间在序列上的差异的影响. 在所采用的实验条件下, Ssh7与双链DNA片段结合时的结合位点大约为6.6 bp, 表观解离常数为(0.7~1.0)×10-7 mol/L. 此外, Ssh7与负超螺旋DNA的结合强于与线性和松弛DNA的结合. 相似文献
6.
嗜热毛壳菌内切β-葡聚糖酶的分离纯化及特性 总被引:5,自引:1,他引:5
探讨了液体发酵嗜热毛壳菌(Chaetomium thermophile)产生的内切β-葡聚糖酶的分离纯化及特性。粗酶液经硫酸铵分级沉淀,DEAE-Seplharose Fast Flow阴离子层析,Pheny1-Sepha-rose疏水层析,Sephacry1 S-100分子筛层析等步骤便可获得凝胶电泳均一的内切β-葡聚糖酶,经12.5%SDS-PAGE和凝胶过滤层析法分离纯化酶蛋白的分子量约为67.8kD的69.8kD。该酶反应的最适温度和pH分别为60℃和4.0-4.5在pH5.0条件下,该酶在60℃下稳定:70℃保温1h后,仍保留30%的活性;在80摄氏度的半衰期为25min,金属离子内切β-葡聚糖酶的活性影响较大,其中Na^ 对酶有激活作用;Fe^2 ,Ag^ ,Cu^2 ,Ba^2 ,Zn^2 等对酶有抑制作用。该酶对结晶纤维素有没水解能力。 相似文献
7.
探讨了液体发酵嗜热毛壳菌(Chaetomium thermophile)产生的内切β葡聚糖酶的分离纯化及特性。粗酶液经硫酸铵分级沉淀、DEAE\|Sepharose Fast Flow阴离子层析、Pheny1\|Sepharose疏水层析、Sephacry1 S\|100分子筛层析等步骤便可获得凝胶电泳均一的内切β\|葡聚糖酶。经125%SDS\|PAGE和凝胶过滤层析法分别测得所分离纯化酶蛋白的分子量约为67.8kD和69.8kD。该酶反应的最适温度和pH分别为60℃和40~45在pH50条件下,该酶在60℃下稳定;70℃保温1h后,仍保留30%的活性;在80℃的半衰期为25min。金属离子对内切β\|葡聚糖酶的活性影响较大,其中Na+对酶有激活作用;Fe2+、Ag+、Cu2+、Ba2+、Zn2+等对酶有抑制作用。该酶对结晶纤维素没有水解能力。 相似文献
8.
嗜热毛壳菌一种β-葡萄糖苷酶的分离纯化及特性 总被引:2,自引:0,他引:2
研究了嗜热毛壳菌Chaetomiumthermophilum液体发酵产生的一种胞外β-葡萄糖苷酶的分离纯化及特性。粗酶液经硫酸铵沉淀、DEAE-SepharoseFastFlow阴离子层析、Phenyl-Sepharose疏水层析、SephacrylS-100分子筛层析等步骤后获得凝胶电泳均一的β-葡萄糖苷酶。经10%SDS-PAGE和凝胶过滤层析方法分别测得该酶的分子量大小约为118.0kDa和120.1kDa。该酶反应的最适温度为70℃,最适pH值为4.0~5.0。有高的热稳定性,在60℃保温1小时酶活性不丧失,在70℃时的半衰期为16min,在90℃保温10min仍具有7.6%的活性。且能在pH4.0~11.0之间保持稳定。金属离子对β-葡萄糖苷酶的活性影响较大,其中Ca2 、Ba2 对酶有激活作用,而Zn2 、Cu2 、Al3 、Ag 、Hg2 对酶有显著的抑制作用。 相似文献
9.
对本室构建的生产海藻糖的基因工程菌株,即来自古细菌芝田硫化叶菌B12的新型α淀粉酶基因的工程菌的表达外源蛋白条件进行宿主菌,培养基,诱导时间,诱导温度和诱导菌浓的起始OD600等条件的优化,发现在宿主菌为大肠杆菌DH5α,培养基为改进的LB,诱导时间4小时,诱导温度41℃,OD600为06~08时,基因工程菌ESBAM中新型α淀粉酶的表达量最高,新型α淀粉酶活力也最高,与重组酶MTSase一起作用底物直链淀粉,经HLPC检测在反应产物中有海藻糖的生成。 相似文献
10.
[目的]研究重组表达的硫矿硫化叶菌P2分子伴侣β亚基体外同源聚合体的结构和生化功能.[方法]利用PCR技术从硫矿硫化叶菌P2的基因组DNA中克隆得到分子伴侣β亚基的基因,将该基因克隆到表达载体pET-21a( )上并在大肠杆菌BL21(DE3)中实现了表达.对纯化后的β亚基单体进行体外聚合,利用透射电镜观察β分子伴侣的结构,并对其促蛋白折叠性质进行了研究.[结果]硫矿硫化叶菌P2分子伴侣β亚基基因在大肠杆菌BL21中实现了高效表达,纯化后的分子伴侣β亚基单体在ATP和Mg2 存在的条件下可自组装形成分子伴侣聚合体.透射电镜观察表明:该β分子伴侣具有Ⅱ型分子伴侣典型的双层面包圈结构,每个环由8个亚基构成.该β分子伴侣具有ATPase活性,最适反应温度为80℃;它不仅能够促进变性的绿色荧光蛋白(GFP)重新折叠,而且还能有效的提高木聚糖酶的热稳定性.[结论]本文根据P2基因组序列分析预测的分子伴侣基因设计引物,克隆表达了硫矿硫化叶菌P2分子伴侣的β亚基,纯化后对其进行体外聚合,透射电镜观察表明该聚合体具有Ⅱ型分子伴侣的经典结构,功能分析表明该β分子伴侣能够在体外促进异源蛋白质的折叠、提高其它酶分子的热稳定性.这为进一步深入研究嗜热古菌耐热抗逆的分子机制,奠定了良好的基础. 相似文献
11.
Purification and characterization of Thermus thermophilus UvrD 总被引:1,自引:0,他引:1
The DNA helicase UvrD (helicase II) protein plays an important role in nucleotide excision repair, mismatch repair, rolling circular plasmid replication, and in DNA replication. A homologue of the Escherichia coli uvrD gene was previously identified in Thermus thermophilus; however, to date, a UvrD helicase has not been purified and characterized from a thermophile. Here we report the purification and characterization of a UvrD protein from Thermus thermophilus HB8. The purified UvrD has a temperature range from 10 degrees to >65 degrees C, with an optimum of 50 degrees C, within the temperature limits of the assay. The enzyme had a requirement for divalent metal ions and nucleoside triphosphates which related to enzyme activity in the order ATP > dATP > dGTP > GTP > CTP > dCTP > UTP. A simple real-time helicase assay was developed that should facilitate detailed kinetic studies of the enzyme. Evaluation of helicase substrates using this assay showed that the enzyme was highly active on a double-stranded DNA with 5' recessed ends in comparison with substrates with 3' recessed or blunt ends, and supports enzyme translocation in a 3'-5' direction relative to the strand bound by the enzyme. 相似文献
12.
C Elie S Salhi J M Rossignol P Forterre A M De Recondo 《Biochimica et biophysica acta》1988,951(2-3):261-267
The archaebacteria constitute a group of prokaryotes with an intermediate phylogenetic position between eukaryotes and eubacteria. The study of their DNA polymerases may provide valuable information about putative evolutionary relationships between prokaryotic and eukaryotic DNA polymerases. As a first step towards this goal, we have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. This enzyme is a monomeric protein of 100 kDa which can catalyze DNA synthesis using either activated calf thymus DNA or oligonucleotide-primed single-stranded DNA as a template. The activity is optimal at 70 degrees C and the enzyme is thermostable up to 80 degrees C; however, it can still polymerize up to 200 nucleotides at 100 degrees C. These remarkable thermophilic properties and thermostability permit examination of the mechanism of DNA synthesis under conditions of decreased stability of the DNA helix. Furthermore, these properties make S. acidocaldarius DNA polymerase a very efficient enzyme to be used in DNA amplification by the recently developed polymerase chain reaction method (PCR) as well as in the Sanger DNA sequencing technique. 相似文献
13.
Characterization of the helicase activity of the Escherichia coli RecBCD enzyme using a novel helicase assay 总被引:14,自引:0,他引:14
We describe an assay to measure the extent of enzymatic unwinding of DNA by a DNA helicase. This assay takes advantage of the quenching of the intrinsic protein fluorescence of Escherichia coli SSB protein upon binding to ssDNA and is used to characterize the DNA unwinding activity of recBCD enzyme. Unwinding in this assay is dependent on the presence of recBCD enzyme and linear dsDNA, is consistent with the known properties of recBCD enzyme, and closely parallels other methods for measuring recBCD enzyme helicase activity. The effects of varying temperature, substrate concentrations, enzyme concentration, and mono- and divalent salt concentrations on the helicase activity of recBCD enzyme were characterized. The apparent Km values for recBCD enzyme helicase activity on linear M13 dsDNA molecules at 25 degrees C are 0.6 nM dsDNA molecules and 130 microM ATP, respectively. The apparent turnover number for unwinding is approximately 15 microM base pairs s-1 (microM recBCD enzyme)-1. When this rate is corrected for the observed stoichiometry of recBCD enzyme binding to dsDNA, kcat for helicase activity corresponds to an unwinding rate of approximately 250 base pairs of DNA s-1 (functional recBCD complex)-1 at 25 degrees C. At 37 degrees C, the apparent Km value for dsDNA molecules was the same as that at 25 degrees C, but the apparent turnover number became 56 microM base pairs s-1 (microM recBCD enzyme)-1 [or 930 base pairs s-1 (functional recBCD complex)-1 when corrected for observed stoichiometry]. With increasing NaCl concentration, kcat peaks at 100 mM, and the apparent Km value for dsDNA increases by 3-fold at 200 mM NaCl. In the presence of 5 mM calcium acetate, the apparent Km value is increased by 3-fold, and kcat decreased by 20-30%. We have also shown that recBCD enzyme molecules are able to catalytically unwind additional dsDNA substrates subsequent to initiation, unwinding, and dissociation from a previous dsDNA molecule. 相似文献
14.
R Meulenberg J T Pronk J Frank W Hazeu P Bos J G Kuenen 《European journal of biochemistry》1992,209(1):367-374
Cell-free extracts of Thiobacillus acidophilus catalysed the quantitative conversion of trithionate (S3O6(2-) to thiosulphate and sulphate. A continuous assay for quantification of experimental results was based on the difference in absorbance between trithionate and thiosulphate at 220 nm. Trithionate hydrolase was purified to near homogeneity from cell-free extracts of T. acidophilus. The molecular masses of the native enzyme and the subunit were 99 kDa (gel filtration) and 34 kDa (SDS/PAGE). The purified enzyme has a pH optimum of 3.5-4.5 and a temperature optimum of 70 degrees C. Enzyme activity was stimulated by sulphate. The stimulation of the enzyme activity by sulphate was half maximal at a concentration of 0.23 M. The Km for trithionate is 70 microM at 30 degrees C and 270 microM at 70 degrees C. Enzyme activity was lost after 36 days at 0 degrees C, 27 days at 70 degrees C; but after 97 days at 30 degrees C, 40% of the initial activity was still present: The enzyme activity was inhibited by mercury chloride, N-ethylmaleimide, thiosulphate and tetrathionate. Tetrathionate S4O6(2-) was not hydrolysed by trithionate hydrolase. 相似文献
15.
Meiosis in Coprinus: characterization and activities of two forms of DNA polymerase during meiotic stages. 总被引:2,自引:1,他引:1
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Two forms of DNA polymerase have been studied in the basidiomycete Coprinus. DNA polymerase from basidiocarp tissues at zygotene-pachytene stage has been purified 3,500-fold and defined as DNA polymerase b by virtue of its insensitivity to N-ethylmaleimide and by its low molecular weight (76,000). This enzyme has optimal activity at pH 7.0 to 7.5, at 200 mM KCl, and at 25 degrees C incubation temperature. It can use polycytidylic acid-oligo(dG)12-18 as template primer in addition to homodeoxypolymers. The DNA polymerase a is mainly produced in the exponentially growing mycelium. It is sensitive to N-ethylmaleimide and has a temperature optimum at 35 degrees C. At the premeiotic S phase, activities from both polymerase a and polymerase b are found in cell-free extracts. The b enzyme is the only DNA polymerase produced during meiotic prophase. Its assayable activity exhibits two peaks, one at premeiotic S stage and one at pachytene. It is possible that DNA polymerase b is responsible for pachytene repairs involved in recombination. 相似文献
16.
We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress. 相似文献
17.
Raffaele Cannio Patrizia Contursi Mosè Rossi Simonetta Bartolucci 《Extremophiles : life under extreme conditions》2001,5(3):153-159
A mutated version of the hygromycin B phosphotransferase (hph(mut)) gene from Escherichia coli, isolated by directed evolution at 75 degrees C in transformants of a thermophilic strain of Sulfolobus solfataricus, was characterized with respect to its genetic stability in both the original mesophilic and the new thermophilic hosts. This gene was demonstrated to be able to express the hygromycin B resistance phenotype and to be steadily maintained and propagated also in other, more thermophilic strains of S. solfataricus, i.e., up to 82 degrees C. Furthermore, it may be transferred to S. solfataricus cells by cotransformation with pKMSD48, another extrachromosomal element derived from the virus SSV1 of Sulfolobus shibatae, without any loss of stability and without affecting the replication and infectivity of this viral DNA. The hph(mut) and the wild-type gene products were expressed at higher levels in E. coli and purified by specific affinity chromatography on immobilized hygromycin B. Comparative characterization revealed that the mutant enzyme had acquired significant thermoresistance and displayed higher thermal activity with augmented catalytic efficiency. 相似文献
18.
Uracil-DNA glycosylase of thermophilic Thermothrix thiopara. 总被引:1,自引:1,他引:0
An activity which released free uracil from dUMP-containing DNA was purified approximately 1,700-fold from extracts of Thermothrix thiopara, the first such activity to be isolated from extremely thermophilic bacteria. The enzyme appeared homogeneous, according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It had a native molecular weight of 26,000 and existed as a monomer protein in water solution. The enzyme had an optimal activity at 70 degrees C, between pH 7.5 and 9.0, and in the presence of 0.2% Triton X-100. It had no cofactor requirement and was not inhibited by EDTA, but it was sensitive to N-ethylmaleimide. The purified enzyme did not contain any nuclease that acted on native or depurinated DNA. The Arrhenius activation energy was 76 kJ/mol between 30 and 50 degrees C and 11 kJ/mol between 50 and 70 degrees C. The rate of heat inactivation of the enzyme followed first-order kinetics with a half-life of 2 min at 70 degrees C. Ammonium sulfate and bovine serum albumin protected the enzyme from heat inactivation. One T. thiopara cell contains enough activity to release about 2 X 10(8) uracil residues from DNA during one generation time at 70 degrees C. 相似文献
19.
A species of DNA polymerase alpha that is active in the ATP(GTP)-dependent conversion of MVM parvovirus single-stranded linear DNA to the duplex replicative form has been purified 4300-fold from Ehrlich ascites mouse tumour cells. The single-stranded----replicative form activity is maintained throughout ammonium sulfate precipitation, DEAE-cellulose, phosphocellulose and hydroxyapatite column chromatography and glycerol gradient sedimentation. Polypeptides with Mr = 230 000, 220 000, 183 000, 157 000, 125 000, 70 000, 65 000, 62 000, 57 000, 53 000 and 48 000 copurify with the single-stranded----replicative form activity, which sediments at approx. 10 S. The Mr = 183 000, 157 000 and 125 000 polypeptides exhibit catalytic activity when assayed in situ following SDS-polyacrylamide gel electrophoresis. The 10 S form of DNA polymerase alpha is functionally distinguishable from an 8.4 S form of the enzyme obtained from the same cells on the basis of single-stranded----replicative form activity. The single-stranded----replicative form activity of the 10 S enzyme is stable at 22 degrees C for up to 3 h, but exhibits a half life of only 5 min at 45 degrees C. 相似文献
20.
Reverse gyrase is the only DNA topoisomerase capable of introducing positive supercoiling into DNA molecules. This unique activity reflects a distinctive arrangement of the protein, which is composed of a topoisomerase IA module fused to a domain containing sequence motives typical of helicases; however, reverse gyrase works neither like a canonical topoisomerase IA nor like a helicase. Extensive genomic analysis has shown that reverse gyrase is present in all organisms living above 70 degrees C and in some of those living at 60- 70 degrees C, but is invariably absent in organisms living at mesophilic temperatures. For its peculiar distribution and biochemical activity, the enzyme has been suggested to play a role in maintenance of genome stability at high temperature. We review here recent phylogenetic, biochemical and structural data on reverse gyrase and discuss the possible role of this enzyme in the biology of hyperthermophilic organisms. 相似文献