肺炎衣原体核糖核酸酶H的功能研究
发布时间：2019-06-15 13:38
【摘要】：核糖核酸酶H (RNase H)能够特异地水解RNA/DNA杂合双链或DNA-RNA-DNA/DNA嵌合型底物中的RNA。根据氨基酸序列及空间结构相似性，RNase H分为两类，1型和2型。其中，1型RNaseH包括细菌的RNase HI及真核生物的RNase H1；2型RNase H包括细菌RNase HII、RNase HIII、古细菌RNase HII和真核生物RNase H2。 RNase HII/H2广泛存在于各种生物体内，但RNase HI/H1和HIII只存留在部分生物体内。目前人们普遍接受的看法是，RNase HI/H1和HIII只能切割含有四个(或更多)核糖核苷酸的DNA-(rN)n-DNA/DNA双链(n≥4)或RNA/DNA杂合链底物，而RNase HII/H2不仅可以切这些底物，还能切割DNA-rN_1-DNA/DNA(rN_1，单个核糖核苷酸)双链。 基因组全序列分析表明，肺炎衣原体没有RNase HI，只有两个2型的RNase H：CpRNase HII和CpRNase HIII，分别由CP0654和CP0782(NCBI序列号)基因编码。我们前期的体外生化研究证实纯化后的CpRNase HII蛋白能切割DNA-rN_1-DNA/DNA底物；而CpRNase HIII可以切RNA/DNA底物。本研究发现CpRNase HIII在锰离子(Mn~（2+）)存在时也能切割DNA-rN_1-DNA/DNA底物。这是RNase H领域中首次报道RNase HIII具有切割DNA-rN_1-DNA/DNA底物的能力。 体外生化实验证实，两种CpRNase H切割DNA-rN_1-DNA/DNA底物时对金属离子的依赖性不同，CpRNase HIII依赖Mn~（2+），而CpRNase HII则偏爱镁离子(Mg2+)且活性会受到Mn~（2+）抑制。进一步研究表明，在切割DNA-rN_1-DNA/DNA底物时，两种酶对反应体系中的镁锰离子波动敏感。另一方面，两种CpRNase H切割其它底物(RNA/DNA杂合链及类似冈崎片段的底物)时酶活性并不会因为镁锰离子波动受到明显影响。这些结果表明镁锰离子水平的变化会抑制一种CpRNase H切割DNA-rN_1-DNA/DNA底物的活性但同时激活另一种CpRNase H的活性。 在细菌体内，我们也证实了上述体外实验的结果。采用基因重组技术构建了三株大肠杆菌rnh突变株，基因改造情况为：LZ1[DY329,ΔrnhA ΔrnhB:: CprnhB]，LZ2[DY329, ΔrnhA:: CprnhC ΔrnhB::CprnhB]，LZ3[DY329, ΔrnhA:: CprnhC ΔrnhB]。其中，CprnhB和CprnhC分别代表两种CpRNase H (HII和HIII)的编码基因；rnhA和rnhB分别表示大肠杆菌RNase HI和HII的编码基因。CpRNase HII遗传互补大肠杆菌RNase H缺失依赖于Mg2+，但培养基中添加0.2mM Mn~（2+）抑制了CpRNase HII的该功能，导致细菌生长缓慢；相反，CpRNase HIII弥补大肠杆菌RNase H缺失则依赖于Mn~（2+）。对大肠杆菌突变株的基因组进行碱敏感性分析发现，，当CpRNase H活性受到抑制而导致细菌生长迟缓时，其基因组对碱非常敏感，表明此时基因组中掺入了大量核糖核苷酸。考虑到体外实验证实CpRNase H酶切RNA/DNA杂合链、类似冈崎片段的底物时不受缓冲液中镁锰离子波动的影响，突变株生长迟缓的主因是体内CpRNase H切割DNA-rN_1-DNA/DNA底物的活性受到抑制，导致基因组中掺入了过多的单个核糖核苷酸；而在生长培养基内添加对应喜好的金属离子则会恢复CpRNase H的活性从而使突变株恢复正常生长。 培养基内添加锰离子影响了细菌体内CpRNase H的活性，这个结果暗示培养基内添加锰时细菌胞内的锰浓度发生了变化，我们提供了相关数据证实这一点。采用含有或不含锰的培养基培养这三株大肠杆菌突变株，用等离子发射光谱(ICP-AES)测定细菌胞内锰离子浓度。结果表明含锰培养基培养的细菌相比不含锰培养基培养的，其胞内锰离子浓度增加了5~14倍，对胞内RNase H活性产生了明显影响，即促进CpRNase HIII活性、抑制CpRNase HII活性。另外，为了验证培养基中添加的锰是否会影响CpRNase H编码基因的表达，我们选择在有锰和无锰时生长有差异的突变株并提取其总RNA进行实时定量PCR。采用管家基因gapA作为内参基因做相对定量分析，结果证实基因表达并未因培养基中添加或缺少锰而有明显差异，表明培养基中的锰影响突变株生长是因为CpRNase H的活性受到抑制，而不是CpRNase H编码基因的表达受阻。 这些实验结果表明，两种CpRNase H在体内是合作互助的关系：在正常情况下由CpRNase HII执行功能，去除基因组中掺入的单个核糖核苷酸；而在离子波动的情况下，比如锰含量较高时，CpRNase HII的活性受到抑制而由CpRNase HIII行使同样的功能。肺炎衣原体采用了两种2型RNase H很可能是因为自身生存环境复杂，在复杂多变的外界环境中两种CpRNase H能够合作、互补，从而使细胞可以维持正常生理代谢。 在证实CpRNase HIII也具有酶切DNA-rN_1-DNA/DNA底物的活性之后，我们进一步研究了CpRNase HIII识别、切割这类底物的结构基础。通过体外生化测活，鉴定突变的氨基酸对该蛋白切割、识别底物的重要性；结合同源模建、分子对接及分子动力学模拟等计算机辅助的方法，阐明了CpRNase HIII识别DNA-rN_1-DNA/DNA底物的机制。CpRNase HIII的“GKG”基序负责识别单个核糖核苷酸，识别方式与RNase HII中“GR (K) G”基序相似，表明CpRNase HIII采纳了与HII相似的底物识别机制。RNase HII/H2识别核糖核苷酸还需要一个高度保守的酪氨酸(Y)，但通过分析氨基酸序列及同源模建得到的蛋白结构模型，发现在CpRNase HIII对应位置上没有这样一个Y残基，通过分子动力学模拟我们发现CpRNase HIII的第94位的丝氨酸(Ser94)与DNA-rN_1-DNA/DNA底物中嵌合的单个核糖核苷酸3′-端的脱氧核糖核苷酸形成稳定氢键，将该脱氧核糖核苷酸拖拽地偏离了核糖核苷酸，同时使DNA双螺旋的局部构象发生了变动。这个举动似乎执行了RNase HII中酪氨酸的功能，援助“GKG”基序准确地识别核糖核苷酸的2′-OH。在分子模拟结果的指导下，我们围绕Ser94进行了一系列生化实验，证明了该Ser对酶活性的重要性。这部分研究结果阐释了CpRNase HIII的底物识别机制。
[Abstract]:The RNase H (RNase H) is capable of specifically hydrolyzing the RNA/ DNA hybrid double-stranded or DNA-RNA-DNA/ DNA chimeric substrate. According to the amino acid sequence and spatial structure similarity, the RNase H is divided into two types: type 1 and type 2. Wherein, the type 1 RNaseH comprises the RNase HI of the bacterium and the RNase H1 of the eukaryote; the type 2 RNase H comprises the bacterial RNase HII, the RNase HIII, the archaea RNase HII and the eukaryote RNase H2. RNase HII/ H2 is widely present in various organisms, but RNase HI/ H1 and HIII remain only in some organisms It is generally accepted that RNase HI/ H1 and HIII can only cut DNA-(rN) n-DNA/ DNA double-stranded (n-4) or RNA/ DNA hybrid chain substrate containing four (or more) ribose nucleic acid, while RNase HII/ H2 can not only cut these substrates, but also cut DNA-rN _ 1-DNA/ DNA (rN _ 1, single ribose nucleic acid) bis The complete sequence analysis of the genome showed that the Chlamydia pneumoniae did not have RNase HI, only two of the two types of RNase H: CpRNase HII and CpRNase HIII, respectively, were based on CP0654 and CP0782 (NCBI serial number). For coding. In-vitro biochemical studies in the early stage confirmed that the purified CpRNase HII protein can cut the DNA-rN _ 1-DNA/ DNA substrate; and CpRNase HIII can cut the RNA/ DN A substrate. The present study found that CpRNase HIII can also cut DNA-rN _ 1-DNA/ DN in the presence of manganese ions (Mn ~ (2 +)) A substrate. This is the first time in the RNase H domain. The RNase HIII has the cleavage DNA-rN _ 1-DNA/ DNA substrate The ability of the two CpRNase H to cut the DNA-rN_1-DNA/ DNA substrate was different when the two CpRNase H cut the DNA-rN_1-DNA/ DNA substrate, and the CpRNase HIII was dependent on Mn ~ (2 +), while the CpRNase HII preferred the magnesium ion (Mg2 +) and the activity was affected by Mn ~ (2 +). 2 +) inhibition. Further studies have shown that, in the cleavage of DNA-rN _ 1-DNA/ DNA substrate, the two enzymes are separated from the magnesium and manganese in the reaction system. On the other hand, the enzyme activity is not affected by the fluctuation of magnesium-manganese ions when two CpRNase H cuts other substrates (the substrate of the RNA/ DNA hybrid chain and the similar Okazaki fragment). These results indicate that a change in the level of magnesium-manganese ions would inhibit the activity of a CpRNase H-cut DNA-rN _ 1-DNA/ DNA substrate, but also activate another CpRNas e H. In the bacterial body, we also confirmed the above The results of in vitro experiments were as follows: The gene recombination technique was used to construct the rnh mutant of Sanzhu. The transformation of the gene was as follows: LZ1[DY329,[rnhA, rnhB:: CprnhB], LZ2[DY329, and rnhA:: CprnhC, rnhB:: CprnhB], LZ3[DY329]: Cprnh: Cprnh C. rnhB], where CprnhB and Cprnhc represent the coding genes of two CpRNase H (HII and HIII), respectively; and rnhA and rnhB represent E. coli RNase HI and H, respectively. The addition of 0.2 mM Mn to (2 +) in the culture medium inhibited the function of the CpRNase HII, resulting in a slow growth of the bacteria; on the contrary, the deletion of the RNase H in the E.coli was dependent on the CpRNase HIII. The analysis of the alkali sensitivity of the genome of the mutant strain of E. coli found that when the activity of the CpRNase H was inhibited and the growth of the bacteria was retarded, its genome was very sensitive to the base, indicating that the genome was incorporated. Large amount of ribose nucleic acid. Considering in vitro experiments that the CpRNase H enzyme digestion RNA/ DNA hybrid chain is confirmed, the substrate with similar Okazaki fragment is not affected by the fluctuation of the magnesium-manganese ions in the buffer solution, the main reason for the growth retardation of the mutant strain is the in vivo CpRNase H cleavage DNA-rN_1-DNA/ DNA substrate, The activity is inhibited, leading to the incorporation of too much of a single ribose nucleic acid in the genome; while adding corresponding preferred metal ions in the growth medium will restore the activity of CpRNase H to make the mutation The addition of manganese ions in the culture medium affected the activity of CpRNase H in the bacteria. The results suggested that the manganese concentration in the bacteria was changed when the manganese was added to the culture medium, and we provided it. This was confirmed by the relevant data. The three strains of E. coli were cultured using a medium containing or without manganese, and measured by plasma emission spectroscopy (ICP-AES). The results showed that the concentration of manganese ion in the cell was increased by 5-14 times compared with that of the culture of the manganese-containing medium, and the activity of the intracellular RNase H was significantly affected, that is to promote the activity of CpRNase HIII and to inhibit the CpR. Nase HII activity. In addition, in order to verify that the added manganese in the medium will affect the expression of the CpRNase H-encoding gene, we choose to grow a mutant strain that is different in the presence of manganese and manganese and to extract its total RNA Real-time quantitative PCR was carried out. The relative quantitative analysis was carried out using the housekeeping gene gapA as the internal reference gene, and the results showed that the expression of the gene was not significantly different from the addition or absence of manganese in the culture medium, indicating that the manganese in the culture medium affected the growth of the mutant strain because of CpRNas. The activity of e H is inhibited, not CpRNase H The expression of the coding gene is blocked. The results of these experiments show that the two kinds of CpRNase H are the relationship of mutual assistance in the body: in the normal case, the function of CpRNase HII is performed to remove the single ribose nucleic acid which is incorporated in the genome, while in the condition of the ion fluctuation If, for example, the manganese content is high, the activity of CpRNase HII is inhibited and the activity of CpRNase HII is inhibited by CpRNase. The same function is exercised by HIII. Two types of RNase H are used by Chlamydia pneumoniae because of their complex living environment, and the two species of CpRNase H can be co-operative and complementary in a complex and changeable environment. The cell can maintain normal physiological metabolism. After confirming that CpRNase HIII also has the activity of the enzyme-cut DNA-rN _ 1-DNA/ DNA substrate, we further studied CpRNase HIII. The structure of this kind of substrate is not cut, and the importance of the substrate is identified by the in vitro biochemical measurement, the amino acid of the mutation is identified and the protein is cut, the importance of the substrate is identified, and the identification of the DNA-rN _ 1 by the CpRNase HIII is clarified by means of computer-aided methods such as the homologous mode construction, the molecular docking and the molecular dynamics simulation. -The mechanism of the DNA/ DNA substrate. The "GKG" motif of the CpRNase HIII was responsible for the identification of a single ribose nucleic acid, similar to the "GR (K) G" motif in the RNase HII, indicating that CpRNase HIII was adopted The substrate recognition mechanism similar to HII. The RNase HII/ H2 recognizes that the ribose nucleic acid also requires a highly conserved tyrosine (Y), but by analyzing the protein structure model obtained by the amino acid sequence and the homologous model, it is found that in the case of CpRNase HIII, There was no such Y residue in position, and by molecular dynamics simulation we found that the 94-bit serine (Ser94) of the CpRNase HIII and the deoxyribose nucleic acid 3--terminal of the DNA-rN _ 1-DNA/ DNA substrate form a stable hydrogen bond, and the deoxyribose core The acid is dragged by the acid of the ribose nucleic acid, while the DNA is allowed to The local conformation of the double helix has changed. This action appears to have performed the function of tyrosine in the RNase HII, and the aid of the "GKG" motif is accurate. In the guidance of the molecular simulation results, we carried out a series of biochemical experiments around Ser94 to prove that The importance of the Ser to the activity of the enzyme is given. The results of this study illustrate the CpRNa
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R374
[Abstract]:The RNase H (RNase H) is capable of specifically hydrolyzing the RNA/ DNA hybrid double-stranded or DNA-RNA-DNA/ DNA chimeric substrate. According to the amino acid sequence and spatial structure similarity, the RNase H is divided into two types: type 1 and type 2. Wherein, the type 1 RNaseH comprises the RNase HI of the bacterium and the RNase H1 of the eukaryote; the type 2 RNase H comprises the bacterial RNase HII, the RNase HIII, the archaea RNase HII and the eukaryote RNase H2. RNase HII/ H2 is widely present in various organisms, but RNase HI/ H1 and HIII remain only in some organisms It is generally accepted that RNase HI/ H1 and HIII can only cut DNA-(rN) n-DNA/ DNA double-stranded (n-4) or RNA/ DNA hybrid chain substrate containing four (or more) ribose nucleic acid, while RNase HII/ H2 can not only cut these substrates, but also cut DNA-rN _ 1-DNA/ DNA (rN _ 1, single ribose nucleic acid) bis The complete sequence analysis of the genome showed that the Chlamydia pneumoniae did not have RNase HI, only two of the two types of RNase H: CpRNase HII and CpRNase HIII, respectively, were based on CP0654 and CP0782 (NCBI serial number). For coding. In-vitro biochemical studies in the early stage confirmed that the purified CpRNase HII protein can cut the DNA-rN _ 1-DNA/ DNA substrate; and CpRNase HIII can cut the RNA/ DN A substrate. The present study found that CpRNase HIII can also cut DNA-rN _ 1-DNA/ DN in the presence of manganese ions (Mn ~ (2 +)) A substrate. This is the first time in the RNase H domain. The RNase HIII has the cleavage DNA-rN _ 1-DNA/ DNA substrate The ability of the two CpRNase H to cut the DNA-rN_1-DNA/ DNA substrate was different when the two CpRNase H cut the DNA-rN_1-DNA/ DNA substrate, and the CpRNase HIII was dependent on Mn ~ (2 +), while the CpRNase HII preferred the magnesium ion (Mg2 +) and the activity was affected by Mn ~ (2 +). 2 +) inhibition. Further studies have shown that, in the cleavage of DNA-rN _ 1-DNA/ DNA substrate, the two enzymes are separated from the magnesium and manganese in the reaction system. On the other hand, the enzyme activity is not affected by the fluctuation of magnesium-manganese ions when two CpRNase H cuts other substrates (the substrate of the RNA/ DNA hybrid chain and the similar Okazaki fragment). These results indicate that a change in the level of magnesium-manganese ions would inhibit the activity of a CpRNase H-cut DNA-rN _ 1-DNA/ DNA substrate, but also activate another CpRNas e H. In the bacterial body, we also confirmed the above The results of in vitro experiments were as follows: The gene recombination technique was used to construct the rnh mutant of Sanzhu. The transformation of the gene was as follows: LZ1[DY329,[rnhA, rnhB:: CprnhB], LZ2[DY329, and rnhA:: CprnhC, rnhB:: CprnhB], LZ3[DY329]: Cprnh: Cprnh C. rnhB], where CprnhB and Cprnhc represent the coding genes of two CpRNase H (HII and HIII), respectively; and rnhA and rnhB represent E. coli RNase HI and H, respectively. The addition of 0.2 mM Mn to (2 +) in the culture medium inhibited the function of the CpRNase HII, resulting in a slow growth of the bacteria; on the contrary, the deletion of the RNase H in the E.coli was dependent on the CpRNase HIII. The analysis of the alkali sensitivity of the genome of the mutant strain of E. coli found that when the activity of the CpRNase H was inhibited and the growth of the bacteria was retarded, its genome was very sensitive to the base, indicating that the genome was incorporated. Large amount of ribose nucleic acid. Considering in vitro experiments that the CpRNase H enzyme digestion RNA/ DNA hybrid chain is confirmed, the substrate with similar Okazaki fragment is not affected by the fluctuation of the magnesium-manganese ions in the buffer solution, the main reason for the growth retardation of the mutant strain is the in vivo CpRNase H cleavage DNA-rN_1-DNA/ DNA substrate, The activity is inhibited, leading to the incorporation of too much of a single ribose nucleic acid in the genome; while adding corresponding preferred metal ions in the growth medium will restore the activity of CpRNase H to make the mutation The addition of manganese ions in the culture medium affected the activity of CpRNase H in the bacteria. The results suggested that the manganese concentration in the bacteria was changed when the manganese was added to the culture medium, and we provided it. This was confirmed by the relevant data. The three strains of E. coli were cultured using a medium containing or without manganese, and measured by plasma emission spectroscopy (ICP-AES). The results showed that the concentration of manganese ion in the cell was increased by 5-14 times compared with that of the culture of the manganese-containing medium, and the activity of the intracellular RNase H was significantly affected, that is to promote the activity of CpRNase HIII and to inhibit the CpR. Nase HII activity. In addition, in order to verify that the added manganese in the medium will affect the expression of the CpRNase H-encoding gene, we choose to grow a mutant strain that is different in the presence of manganese and manganese and to extract its total RNA Real-time quantitative PCR was carried out. The relative quantitative analysis was carried out using the housekeeping gene gapA as the internal reference gene, and the results showed that the expression of the gene was not significantly different from the addition or absence of manganese in the culture medium, indicating that the manganese in the culture medium affected the growth of the mutant strain because of CpRNas. The activity of e H is inhibited, not CpRNase H The expression of the coding gene is blocked. The results of these experiments show that the two kinds of CpRNase H are the relationship of mutual assistance in the body: in the normal case, the function of CpRNase HII is performed to remove the single ribose nucleic acid which is incorporated in the genome, while in the condition of the ion fluctuation If, for example, the manganese content is high, the activity of CpRNase HII is inhibited and the activity of CpRNase HII is inhibited by CpRNase. The same function is exercised by HIII. Two types of RNase H are used by Chlamydia pneumoniae because of their complex living environment, and the two species of CpRNase H can be co-operative and complementary in a complex and changeable environment. The cell can maintain normal physiological metabolism. After confirming that CpRNase HIII also has the activity of the enzyme-cut DNA-rN _ 1-DNA/ DNA substrate, we further studied CpRNase HIII. The structure of this kind of substrate is not cut, and the importance of the substrate is identified by the in vitro biochemical measurement, the amino acid of the mutation is identified and the protein is cut, the importance of the substrate is identified, and the identification of the DNA-rN _ 1 by the CpRNase HIII is clarified by means of computer-aided methods such as the homologous mode construction, the molecular docking and the molecular dynamics simulation. -The mechanism of the DNA/ DNA substrate. The "GKG" motif of the CpRNase HIII was responsible for the identification of a single ribose nucleic acid, similar to the "GR (K) G" motif in the RNase HII, indicating that CpRNase HIII was adopted The substrate recognition mechanism similar to HII. The RNase HII/ H2 recognizes that the ribose nucleic acid also requires a highly conserved tyrosine (Y), but by analyzing the protein structure model obtained by the amino acid sequence and the homologous model, it is found that in the case of CpRNase HIII, There was no such Y residue in position, and by molecular dynamics simulation we found that the 94-bit serine (Ser94) of the CpRNase HIII and the deoxyribose nucleic acid 3--terminal of the DNA-rN _ 1-DNA/ DNA substrate form a stable hydrogen bond, and the deoxyribose core The acid is dragged by the acid of the ribose nucleic acid, while the DNA is allowed to The local conformation of the double helix has changed. This action appears to have performed the function of tyrosine in the RNase HII, and the aid of the "GKG" motif is accurate. In the guidance of the molecular simulation results, we carried out a series of biochemical experiments around Ser94 to prove that The importance of the Ser to the activity of the enzyme is given. The results of this study illustrate the CpRNa
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R374
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5 倪安平;崔京涛;汪晓巍;;4种国产大环内酯类抗菌药物体外抗沙眼衣原体和肺炎衣原体作用的研究[A];中华医学会第七次全国检验医学学术会议资料汇编[C];2008年
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7 苏世斌;林勤益;郭浩然;;台湾成年人肺炎衣原体和新陈代谢症候群之相关研究[A];第七届海峡两岸心血管科学研讨会论文集[C];2009年
8 杨玲;吴移谋;周洲;邓仲良;刘R
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