鸟嘌呤碱基与羟基自由基反应的密度泛函理论

羟基自由基 (·OH)进攻嘌呤碱基是破坏核酸造成DNA断链损伤的重要原因之一 .采用密度泛函 (DFT)理论中B3LYP方法在 6— 31G基组水平上对鸟嘌呤 (G)受羟基自由基进攻形成的各种可能产物自由基进行几何全优化 .根据总能量、键长和自旋密度的计算结果 ,从理论上确认了C 5和C 8位加成机制 .得产物自由基G5OH·、G8OH· ,且G5OH·易与N 11位H脱水得一个更稳定的产物自由基 ,而G8OH·不易发生开环反应 ,得到与实验一致的结论 .这些稳定自由基的形成造成DNA断链损伤     

谈谈DNA的碱基配对

在高中“生物”课讲脱氧核糖核酸(DNA)的分子结构,介绍碱基互补配对原则时,学生常会向老师提出:为什么腺嘌呤(A)一定与胸腺嘧啶(T)配对,鸟嘌呤(G)一定与胞嘧啶(C)配对。从如下几方面解释可较圆满地回答这个问题: 1.每种生物双链DNA的四种碱基都是腺嘌呤(A)的百分比等于胸腺嘧啶(T)的百分比;鸟嘌呤(G)的百分比等于胞嘧啶(C)的百分比。     

碱金属离子——Na~ 与 DNA 中碱基,配对碱基,双联体↓■↑、↓■↑的相互作用

本文根据分子中原子和离子之间非键相互作用的半经验势函数,并应用BFGS变尺度法进行最优化处理而研究Na~ 与脱氧核糖核酸(DNA)中碱基(胞嘧啶(C)、鸟嘌呤(G)、腺嘌呤(A)、胸腺嘧啶(T)),配对碱基G—C、A—T 碱基对,螺旋双联体↓■↑、↓■↑等的相互作用,得到了它们各自的最优配位模式。     

脂质过氧化引起的DNA损伤研究进展

脂质过氧化可以引起各种碱基损伤、DNA链断裂和各种荧光产物生成,并对DNA分子鸟嘌呤碱基具有选择性损伤.过渡金属离子可以明显加深脂质过氧化对DNA的损伤程度.多种抗氧化剂、活性氧自由基清除剂对脂质过氧化引起的DNA损伤有一定程度的保护作用.具有致突、致癌作用的8－羟基鸟嘌呤已经观察到．脂质过氧化的致突变、致癌变作用机制引起了人们的极大兴趣．     

DNA氧化损伤8-羟鸟嘌呤与肿瘤的发生发展

有氧代谢有机体细胞无法避免活性氧(reactive oxygen species, ROS)的伤害。ROS会造成多种形式的DNA损伤,其中鸟嘌呤G的氧化产物8-羟鸟嘌呤(8-oxoG)是频度最高的一种DNA氧化损伤,由特异性的糖苷酶OGG1识别并开启碱基切除修复通路完成修复。8-oxoG如果没有及时修复,可能会在复制的过程中引入G:C配对到T:A配对的碱基颠换突变。因此8-oxoG的积累或OGG1修复功能异常被认为会影响基因功能,进而导致肿瘤或衰老相关疾病的发生,然而直接实验证据却极为有限。近年来一系列研究表明,8-oxoG倾向于产生在基因的调控区,在这种情形下,8-oxoG可视为一种表观遗传学修饰,而OGG1则是这一信息的特异性读取者,OGG1对底物的识别、结合或切除会引发DNA构象或组蛋白修饰的改变,进而引起基因表达的上调或下调。因此,除了潜在的遗传毒性,鸟嘌呤氧化损伤与肿瘤的关联与其通过表观遗传学机制引发基因表达的异常密切相关。本文对8-oxoG及修复酶OGG1与肿瘤发生发展的关联机制进行了分析与总结,旨在提示研究人员从新的视角解读DNA氧化损伤与肿瘤的关系,并为肿瘤的治疗提供新...     

生物工程技术讲座(九)

双链的DNA分子是由一条单链上的碱基与另一条单链上碱基相配对组成。分子中的碱基腺嘌呤(A)和胸腺嘧啶(T)间有两处,鸟嘌呤(G)和胞嘧啶(C)间有三处通过氢(H)结合配对,形成双链间的对应互补。由H在双链分子间形成的结合虽稳定,但是可逆的即经加热或碱     

《生物工程讲座》名词解释(Ⅰ-Ⅳ讲)

1.bp碱基对。DNA长度的单位。1000bp=1kb。 2.kp千碱基对。参见bp。 3.A,G,T,C DNA双链上四种脱氧核苷酸(或其碱基)的符号,A为脱氧腺嘌呤核苷酸,G为脱氧鸟便嘌呤核苷酸,T为脱氧胸腺嘧啶核苷酸,C为脱氧胞嘧啶核苷酸。在双链上。A恒与T配对,G恒与C配对。在RNA链上,四种核苷酸的符号为A,G,u,c,相应为腺嘌呤核苷酸,鸟便嘌呤核苷酸,尿嘧啶核苷酸和胞嘧啶核苷酸。     

κ-卡拉胶琥珀酰基化衍生物的抗氧化活性研究

对κ-卡拉胶进行酸降解得到三种卡拉胶低聚糖,并进一步琥珀酰基化得到分子量分别为2720、4000和5960的κ-卡拉胶琥珀酰衍生物(A、B和C)。对产物进行FT-IR表征,并测得其琥珀酰基取代度(DS)分别为0.61、0.29和0.83。检测了三种κ-卡拉胶琥珀酰衍生物对超氧阴离子自由基O2.-、DPPH自由基、羟基自由基.OH以及过氧化氢的清除活性。结果表明:随着取代度的增加,其清除超氧阴离子自由基O2.-和DPPH自由基的能力增强;随着分子量的增加,其清除羟基自由基.OH和过氧化氢的能力增强。这可能与衍生物的羟基含量、取代基团的性质以及取代度等因素有关。     

限制性内切酶 Sau3AI 的提取与纯化

限制性内切酶Sau3AI是分子生物学与遗传工程研究的重要工具酶之一。它能确定DNA复制的起点或终点的位置,确定RNA在DNA上的转录位置,分析DNA中脱氧核苷酸序列的排列以及基因的分离等。 J.S.Sassenbach等人首先从StaPhylococcus aureus 3A(1)中提取限制性内切酶Sau3AJ,其切割序列为:5′G—A—T—C—3′3′—C—T—G—5″它除了对腺嘌呤的甲基化作用不敏感之外,是MboI的异源同功酶。我们参考J.S.Sussenbach等人的方法,并做了适当的改进,因而获得了较高纯度的Sau3AI核酶内切酶。一、材料与试剂 1、菌种:Staphylococcus aureus(本所保藏,     

通过DNA改组技术获得高活性β-葡萄糖苷酸酶

β 葡萄糖苷酸酶是在植物转基因中广泛应用的报告基因 .以质粒pBI12 1中的GUS基因为基础 ,利用DNA改组方法 ,经DNaseⅠ降解 ,PrimerlessPCR ,PrimerPCR对GUS基因进行了突变和改组 ,然后将改组的GUS基因连接到原核表达载体pG2 5 1中 ,构建了库容为 10 8的突变体库 .经过活性的筛选 ,得到活性提高的克隆 ,再以此为基础 ,经过新的改组、筛选得到活性大幅度提高的克隆GUS2 4 .基因测序显示 ,GUS2 4与GUS基因之间的同源性为 99 7% ,共有 6个核苷酸位点发生了改变 ,分别是 :379位的A突变为G ,396位的T突变为C ,711位的G突变为A ,95 8位T突变为C ,990位的T突变为C ,1649位的A突变为G .核苷酸序列推导的氨基酸序列显示 ,3个氨基酸发生了突变 ,12 7位的Ser突变为Gly ,32 0位的Trp突变为Arg ,5 5 0位的Asn突变为Ser.X gluc染色检测和荧光测活结果显示GUS2 4基因表达的 β 葡萄糖苷酸酶基较GUS基因表达产物活性提高 3倍     

A conserved base-pair between tRNA and 23 S rRNA in the peptidyl transferase center is important for peptide release

The 3' terminus of tRNAs has the universally conserved bases C74C75A76 that interact with the ribosomal large subunit. In the ribosomal P site, bases C74 and C75 of tRNA, form Watson-Crick base-pairs with G2252 and G2251, respectively, present in the conserved P-loop of 23 S rRNA. Previous studies have suggested that the G2252-C74 base-pair is important for peptide bond formation. Using a pure population of mutant ribosomes, we analyzed the precise role of this base-pair in peptide bond formation, elongation factor G-dependent translocation, and peptide release by release factor 1. Surprisingly, our results show that the G2252-C74 base-pair is not essential for peptide bond formation with intact aminoacyl tRNAs as substrates and for EF-G catalyzed translocation. Interestingly, however, peptide release was reduced substantially when base-pair formation between G2252 and C74 of P site tRNA was disrupted, indicating that this conserved base-pair plays an important role in ester bond hydrolysis during translation termination.     

Fibre X-Ray and Conformational Study of the Binding of Metal Ions on DNA

Abstract

Results obtained from X-ray diffraction as well as from conformational analysis of Ag-DNA fibres are presented. For small percentages of Ag⁺ bound and high humidity, the B-DNA form is maintained. As the percentage of Ag⁺ is increased, the helical parameters of the B-DNA are modified. These modifications are directly related to the percentage of G—C bases. The periodicity of the DNA fibres are perturbed as Ag⁺ is mainly bound to G—C pairs and, thus, only the equatorial diffracted intensities can be compared to values calculated from molecular models. It is shown, by this way, that the first binding site is located on N7 of G. A second site is situated between N3 and N1 of the G—C pair, at the place of a hydrogen bond. A molecular model of the Ag-DNA complex is proposed and shown to be in agreement with experimental data. Results obtained allow to get some information on the binding of other ions such as Cu²⁺ and Hg²⁺ which give very little modification of the fibre X-ray patterns.     

Interaction between the two conserved single-stranded regions at the decoding site of small subunit ribosomal RNA is essential for ribosome function.

Formation of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, is essential for ribosome function. It was previously reported that mutation of G1401 inactivated all in vitro functions of the ribosome [Cunningham et al. (1992) Biochemistry 31, 7629-7637]. Here we show that mutation of C1501 to G was equally inactivating but that the double mutant C1401:G1501 with the base pair reversed had virtually full activity for tRNA binding to the P, A, and I sites and for peptide bond formation. Initiation-dependent formation of the first peptide bond remained 70-85% inhibited, despite full 70S initiation complex formation ability as evidenced by the ability to form fMET-puromycin. These results suggest that the defect in formation of the first peptide bond lies in filling the initial A site, Ai, rather than the subsequent elongation A sites, Ae. An increased mobility around the anticodon was detected by UV cross-linking of the anticodon of P-site-bound tRNA to C1399 as well as to the expected C1400. These findings provide the first experimental evidence for the existence of the G1401:C1501 base pair and show that this base pair, located at the decoding site, is essential for function. The structural implications of tertiary base pair formation are discussed.     

The strength of dehalogenase-substrate hydrogen bonding correlates with the rate of Meisenheimer intermediate formation

4-Chlorobenzoyl-coenzyme A (4-CBA-CoA) dehalogenase catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to 4-hydroxybenzoyl-CoA by using an active site aspartate as the nucleophile. Formation of the corresponding Meisenheimer complex (EMc) is followed by chloride ion expulsion which forms the arylated intermediate (EAr). This is then hydrolyzed to the product. In this paper, we explore the relationship between active site polarizing forces acting on the benzoyl carbonyl and the rate of formation of the Meisenheimer complex. The polarizing forces at the C[double bond]O group were modulated by introducing site-selected mutations (A112V, Y65D, G113A, G113S, G113N, and F64P), near the C[double bond]O binding site. Using either the substrate, 4-CBA-CoA, or the substrate analogue, 4-methylbenzoyl-CoA (4-MBA-CoA), Raman difference spectroscopy provided the position of the C[double bond]O stretching frequency (nu(C)[double bond](O)) for a total of 10 enzyme-ligand complexes. In turn, the values of the C[double bond]O frequencies could be converted to differences in effective hydrogen bonding strengths between members of the series, based on earlier model studies [Clarkson, J., Tonge, P. J., Taylor, K. L., Dunaway-Mariano, D., and Carey, P. (1997) Biochemistry 36, 10192-10199]. Catalysis in the F64P, G113A, G113S, and G113N dehalogenase mutants was very slow with k(cat) values ranging from 8 x 10(-3) to 7.6 x 10(-6) s(-1). The EAr intermediate did not accumulate to a detectable level on these enzymes during a single turnover. Catalysis in the Y65D and A112V dehalogenase mutants were almost as efficient as catalysis in wild-type dehalogenase with k(cat) values of 0.1-0.6 s(-1). In wild-type dehalogenase, 22% of the bound substrate accumulated as the EAr intermediate during a single turnover (k(obs) for EAr formation = 24 s(-(1)); in the Y65D mutant, the level of accumulation is 17% (k(obs) for EAr formation = 3 s(-1)), and in the A112V mutant, the level is 23% (k(obs) for EAr formation = 17 s(-1)). The k(obs) for EAr formation in wild-type dehalogenase and the more active dehalogenase mutants (Y65D and A112V) was taken to be an estimate of the k for EMc formation, and the k(obs) for EP formation in a single turnover was taken to be an estimate of the k for EMc formation in the severely impaired mutants (F64P, G113A, G113S, and G113N). A plot of the log k(obs) for EMc formation versus the C[double bond]O stretching frequency of bound 4-CBA-CoA (or 4-MBA-CoA) is a straight line (R(2) = 0.9584). Throughout the series, nu(C)[double bond](O) varied by 61 cm(-1), corresponding to the change in hydrogen bonding enthalpy of 67 kJ/mol. The results show that changes in polarizing forces at the benzoyl carbonyl are transmitted to the benzoyl (4) position and correlate with the rate of aromatic nucleophilic addition five chemical bonds away. Interestingly, the relationship between effective polarizing forces and reactivity seen here for dehalogenase is similar to that reported for the addition-elimination reaction involving the hydrolysis of a series of acyl serine proteases.     

Disulfide bond formation is a determinant of glycosylation site usage in the hemagglutinin-neuraminidase glycoprotein of Newcastle disease virus.

Determinants of glycosylation site usage were explored by using the hemagglutinin-neuraminidase (HN) glycoprotein of the paramyxovirus Newcastle disease virus. The amino acid sequence of the HN protein, a type II glycoprotein, has six N-linked glycosylation addition sites, G1 to G6, two of which, G5 and G6, are not used for the addition of carbohydrate (L. McGinnes and T. Morrison, Virology 212:398-410, 1995). The sequence of this protein also has 13 cysteine residues in the ectodomain (C2 to C14). Mutation of either cysteine 13 or cysteine 14 resulted in the addition of another oligosaccharide chain to the protein. These cysteine residues flank the normally unused G6 glycosylation addition site, and mutation of the G6 site eliminated the extra glycosylation found in the cysteine mutants. These results suggested that failure to form an intramolecular disulfide bond resulted in the usage of a normally unused glycosylation site. This conclusion was confirmed by preventing cotranslational disulfide bond formation in cells by using dithiothreitol. Under these conditions, the wild-type protein acquired extra glycosylation, which was eliminated by mutation of the G6 site. These results suggest that localized folding events on the nascent chain, such as disulfide bond formation, which block access to the oligosaccharyl transferase are a determinant of glycosylation site usage.     

A novel nucleotide excision repair for the conversion of an A/G mismatch to C/G base pair in E. coli

A protein that binds specifically to A/G mismatches has been detected in E. coli by a gel electrophoresis DNA binding assay. A specific endonuclease is associated with the A/G mismatch-binding protein through two chromatographic steps. The endonuclease is specific for A/G-containing DNA fragments and has no cleavage activity on DNA containing the other seven possible mispairs or homoduplex DNA. The endonuclease simultaneously makes incisions at the first phosphodiester bond 3' to and the second phosphodiester bond 5' to the dA of the A/G mismatch. No incision site was detected on the other strand. These results are consistent with the unidirectional A to C conversion and short repair tract of a novel dam- and mutHLS-independent A/G repair pathway we have recently described. A nucleotide excision repair model is proposed for the conversion of an A/G mismatch to a C/G base pair.     

An engineered disulfide bond between residues 69 and 238 in extended-spectrum beta-lactamase Toho-1 reduces its activity toward third-generation cephalosporins

Previous crystallographic structural analysis of extended-spectrum beta-lactamase Toho-1 predicted that the high flexibility of beta-strand B3, the region that contains a conserved KTG motif and forms one wall of the substrate-binding site, could be one of the key features contributing to Toho-1 activity toward third-generation cephalosporins. To investigate whether this possible flexibility really affects the substrate profile of this enzyme, two Toho-1 mutants have been produced, G238C and G238C/G239in, in which the glycine residue at position 238 was replaced with a cysteine and an additional glycine residue was inserted. Our intent was to introduce a disulfide bond between the cysteine residues at positions 69 and 238, and thus to lock the position of beta-strand B3. The results of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) titration indicated formation of a new disulfide bridge in the G238C mutant, although disulfide bond formation was not confirmed in the G238C/G239in mutant. Kinetic analysis showed that the activity of the G238C mutant decreased drastically against third-generation cephalosporins, while its catalytic efficiency against penicillins and first-generation cephalosporins was almost identical to that of the wild-type enzyme. This result was consistent with the prediction that flexibility in beta-strand B3 was critical for activity against third-generation cephalosporins in Toho-1. Furthermore, we have determined the crystal structure of the G238C mutant enzyme to analyze the structural changes in detail. The structural model clearly shows the introduction of a new disulfide bridge and that there is no appreciable difference between the overall structures of the wild-type enzyme and the G238C mutant, although the introduced disulfide bond slightly influenced the positions of Ser237 on beta-strand B3 and Asn170 on the Omega loop. The results of our kinetic and structural analyses suggest that the flexibility of beta-strand B3, as well as the positions of Ser237 and the Omega loop, is critical for the substrate specificity expansion of Toho-1.     

Mechanisms on inhibition of polyethylene electrical tree aging: a theoretical study

A theoretical investigation is completed on inhibition mechanism of polyethylene electrical tree aging. Foremost it elucidates that it is one of the important factors for inhibiting initiation and propagation of polyethylene electrical tree through keto-enol tautomerism of acetophenone and its analogues. Geometries of the keto tautomer and the enol tautomer of acetophenone and its analogues, and its transition states are optimized at the B3LYP/6?311+G(d,p) level, the harmonic vibration frequencies of the equilibrium geometries are calculated at the same level. The minimum energy path (MEP) is obtained by the intrinsic reaction coordinate (IRC) theory at the same level. The calculated results show that the energy barriers of keto-enol tautomerism of acetophenone and its analogues are smaller than the average C—C bond energy of polyethylene, the acetophenone and its analogues adulterated in polyethylene composites can improve the strength of alternate current puncture that PE can endure as well as inhibit polyethylene electrical tree from initiation and propagation.     

Comparison of base specificity and other enzymatic properties of two protozoan ribonucleases from Physarum polycephalum and Dictyostelium discoideum

Base specificity and other enzymatic properties of two protozoan RNases, RNase Phyb from a true slime mold (Physarum polycephalum) and RNase DdI from a cellular slime mold (Dictyostelium discoideum), were compared. These two RNases have high amino acid sequence similarity (83 amino acid residues, 46%). The base specificities of two base recognition sites, The B1 site (base recognition site for the base at 5'-side of scissile phosphodiester bond) and the B2 site (base recognition site for the base at 3'-side of the scissile bond) of the both enzymes were estimated by the rates of hydrolysis of 16 dinucleoside phosphates. The base specificities estimated of B1 and B2 sites of RNase Phyb and RNase DdI were A, G, U > C and A > or = G > C > U, and A > or = G, U > C and G > U > A, C, respectively. The base specificities estimated from the depolymerization of homopolynucleotides and those from the releases of four mononucleotides upon digestion of RNA coincided well with those of the B2 sites of both enzymes. Thus, in these enzymes, the contribution of the B2 site to base specificity seems to be larger than that of the B1 site. pH-stability, optimum temperature, and temperature stability, of both enzymes are discussed considering that RNase Phyb has one disulfide bridge deleted, compared to the RNase DdI with four disulfide bridges.