肺炎衣原體核糖核酸酶H的功能研究
發(fā)布時間：2019-06-15 13:38
【摘要】：核糖核酸酶H (RNase H)能夠特異地水解RNA/DNA雜合雙鏈或DNA-RNA-DNA/DNA嵌合型底物中的RNA。根據(jù)氨基酸序列及空間結(jié)構(gòu)相似性，RNase H分為兩類，1型和2型。其中，1型RNaseH包括細菌的RNase HI及真核生物的RNase H1；2型RNase H包括細菌RNase HII、RNase HIII、古細菌RNase HII和真核生物RNase H2。 RNase HII/H2廣泛存在于各種生物體內(nèi)，但RNase HI/H1和HIII只存留在部分生物體內(nèi)。目前人們普遍接受的看法是，RNase HI/H1和HIII只能切割含有四個(或更多)核糖核苷酸的DNA-(rN)n-DNA/DNA雙鏈(n≥4)或RNA/DNA雜合鏈底物，而RNase HII/H2不僅可以切這些底物，還能切割DNA-rN_1-DNA/DNA(rN_1，單個核糖核苷酸)雙鏈。 基因組全序列分析表明，肺炎衣原體沒有RNase HI，只有兩個2型的RNase H：CpRNase HII和CpRNase HIII，分別由CP0654和CP0782(NCBI序列號)基因編碼。我們前期的體外生化研究證實純化后的CpRNase HII蛋白能切割DNA-rN_1-DNA/DNA底物；而CpRNase HIII可以切RNA/DNA底物。本研究發(fā)現(xiàn)CpRNase HIII在錳離子(Mn~（2+）)存在時也能切割DNA-rN_1-DNA/DNA底物。這是RNase H領域中首次報道RNase HIII具有切割DNA-rN_1-DNA/DNA底物的能力。 體外生化實驗證實，兩種CpRNase H切割DNA-rN_1-DNA/DNA底物時對金屬離子的依賴性不同，CpRNase HIII依賴Mn~（2+），而CpRNase HII則偏愛鎂離子(Mg2+)且活性會受到Mn~（2+）抑制。進一步研究表明，在切割DNA-rN_1-DNA/DNA底物時，兩種酶對反應體系中的鎂錳離子波動敏感。另一方面，兩種CpRNase H切割其它底物(RNA/DNA雜合鏈及類似岡崎片段的底物)時酶活性并不會因為鎂錳離子波動受到明顯影響。這些結(jié)果表明鎂錳離子水平的變化會抑制一種CpRNase H切割DNA-rN_1-DNA/DNA底物的活性但同時激活另一種CpRNase H的活性。 在細菌體內(nèi)，我們也證實了上述體外實驗的結(jié)果。采用基因重組技術(shù)構(gòu)建了三株大腸桿菌rnh突變株，基因改造情況為：LZ1[DY329,ΔrnhA ΔrnhB:: CprnhB]，LZ2[DY329, ΔrnhA:: CprnhC ΔrnhB::CprnhB]，LZ3[DY329, ΔrnhA:: CprnhC ΔrnhB]。其中，CprnhB和CprnhC分別代表兩種CpRNase H (HII和HIII)的編碼基因；rnhA和rnhB分別表示大腸桿菌RNase HI和HII的編碼基因。CpRNase HII遺傳互補大腸桿菌RNase H缺失依賴于Mg2+，但培養(yǎng)基中添加0.2mM Mn~（2+）抑制了CpRNase HII的該功能，導致細菌生長緩慢；相反，CpRNase HIII彌補大腸桿菌RNase H缺失則依賴于Mn~（2+）。對大腸桿菌突變株的基因組進行堿敏感性分析發(fā)現(xiàn)，，當CpRNase H活性受到抑制而導致細菌生長遲緩時，其基因組對堿非常敏感，表明此時基因組中摻入了大量核糖核苷酸。考慮到體外實驗證實CpRNase H酶切RNA/DNA雜合鏈、類似岡崎片段的底物時不受緩沖液中鎂錳離子波動的影響，突變株生長遲緩的主因是體內(nèi)CpRNase H切割DNA-rN_1-DNA/DNA底物的活性受到抑制，導致基因組中摻入了過多的單個核糖核苷酸；而在生長培養(yǎng)基內(nèi)添加對應喜好的金屬離子則會恢復CpRNase H的活性從而使突變株恢復正常生長。 培養(yǎng)基內(nèi)添加錳離子影響了細菌體內(nèi)CpRNase H的活性，這個結(jié)果暗示培養(yǎng)基內(nèi)添加錳時細菌胞內(nèi)的錳濃度發(fā)生了變化，我們提供了相關(guān)數(shù)據(jù)證實這一點。采用含有或不含錳的培養(yǎng)基培養(yǎng)這三株大腸桿菌突變株，用等離子發(fā)射光譜(ICP-AES)測定細菌胞內(nèi)錳離子濃度。結(jié)果表明含錳培養(yǎng)基培養(yǎng)的細菌相比不含錳培養(yǎng)基培養(yǎng)的，其胞內(nèi)錳離子濃度增加了5~14倍，對胞內(nèi)RNase H活性產(chǎn)生了明顯影響，即促進CpRNase HIII活性、抑制CpRNase HII活性。另外，為了驗證培養(yǎng)基中添加的錳是否會影響CpRNase H編碼基因的表達，我們選擇在有錳和無錳時生長有差異的突變株并提取其總RNA進行實時定量PCR。采用管家基因gapA作為內(nèi)參基因做相對定量分析，結(jié)果證實基因表達并未因培養(yǎng)基中添加或缺少錳而有明顯差異，表明培養(yǎng)基中的錳影響突變株生長是因為CpRNase H的活性受到抑制，而不是CpRNase H編碼基因的表達受阻。 這些實驗結(jié)果表明，兩種CpRNase H在體內(nèi)是合作互助的關(guān)系：在正常情況下由CpRNase HII執(zhí)行功能，去除基因組中摻入的單個核糖核苷酸；而在離子波動的情況下，比如錳含量較高時，CpRNase HII的活性受到抑制而由CpRNase HIII行使同樣的功能。肺炎衣原體采用了兩種2型RNase H很可能是因為自身生存環(huán)境復雜，在復雜多變的外界環(huán)境中兩種CpRNase H能夠合作、互補，從而使細胞可以維持正常生理代謝。 在證實CpRNase HIII也具有酶切DNA-rN_1-DNA/DNA底物的活性之后，我們進一步研究了CpRNase HIII識別、切割這類底物的結(jié)構(gòu)基礎。通過體外生化測活，鑒定突變的氨基酸對該蛋白切割、識別底物的重要性；結(jié)合同源模建、分子對接及分子動力學模擬等計算機輔助的方法，闡明了CpRNase HIII識別DNA-rN_1-DNA/DNA底物的機制。CpRNase HIII的“GKG”基序負責識別單個核糖核苷酸，識別方式與RNase HII中“GR (K) G”基序相似，表明CpRNase HIII采納了與HII相似的底物識別機制。RNase HII/H2識別核糖核苷酸還需要一個高度保守的酪氨酸(Y)，但通過分析氨基酸序列及同源模建得到的蛋白結(jié)構(gòu)模型，發(fā)現(xiàn)在CpRNase HIII對應位置上沒有這樣一個Y殘基，通過分子動力學模擬我們發(fā)現(xiàn)CpRNase HIII的第94位的絲氨酸(Ser94)與DNA-rN_1-DNA/DNA底物中嵌合的單個核糖核苷酸3′-端的脫氧核糖核苷酸形成穩(wěn)定氫鍵，將該脫氧核糖核苷酸拖拽地偏離了核糖核苷酸，同時使DNA雙螺旋的局部構(gòu)象發(fā)生了變動。這個舉動似乎執(zhí)行了RNase HII中酪氨酸的功能，援助“GKG”基序準確地識別核糖核苷酸的2′-OH。在分子模擬結(jié)果的指導下，我們圍繞Ser94進行了一系列生化實驗，證明了該Ser對酶活性的重要性。這部分研究結(jié)果闡釋了CpRNase HIII的底物識別機制。
[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
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：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
【學位授予單位】：上海交通大學
【學位級別】：博士
【學位授予年份】：2012
【分類號】：R374
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