假单胞菌Pseudomonas fluorescens Pf0-1中转录调控因子SpfB负调控DNA磷硫酰化修饰（英文）

【目的】DNA磷硫酰化修饰是DNA骨架上非桥接的氧原子以序列选择性和R-构型被硫取代的一种新型DNA修饰。目前,磷硫酰化修饰在多种细菌、古生菌以及人类致病菌中多有发现,但其分子调控机制尚不清楚。为了全面解析磷硫酰化修饰的调控机制,本文选择荧光假单胞菌Pf0-1为研究对象,开展了其DNA磷硫酰化修饰的调控机制研究。【方法】首先,构建了spfB基因缺失和回补菌株,使用碘能特异性断裂磷硫酰化修饰DNA的方法,研究了该基因缺失对修饰表型的影响。利用cDNA在相邻同方向的基因间隔区进行PCR,确定了磷硫酰化修饰基因簇spf BCDE内的共转录单元。通过荧光定量RT-PCR,分析了spfB基因缺失突变株中磷硫酰化修饰基因的转录量。利用异源表达并纯化得到的重组蛋白SpfB进行了体外功能研究。通过EMSA实验,验证了SpfB蛋白具有与spfB启动子序列结合活性。通过DNase I footprinting实验,精确定位了Spf B蛋白与DNA结合序列。【结果】spf B基因的缺失加剧了磷硫酰化修饰DNA断裂所致电泳条带弥散的表型,spf B基因的回补能够恢复该表型,证明spf B基因负调控磷硫酰化修饰。鉴定了spf基因簇中只含有1个共转录单元,且该共转录单元在?spfB突变株中转录水平明显上升。通过EMSA和DNase I footprint实验,检测了SpfB蛋白与磷硫酰化修饰基因spf BCDE的启动子区域5′-TGTTTGT-3′相结合。【结论】SpfB作为转录调控因子负调控磷硫酰化修饰基因spf BCDE的表达,为解析磷硫酰化修饰的调控机制和全面理解基因组上的部分修饰特征奠定了基础。     

假单胞菌Pseudomonas fluorescens Pf0-1中转录调控因子SpfB负调控DNA磷硫酰化修饰

[目的]DNA磷硫酰化修饰是DNA骨架上非桥接的氧原子以序列选择性和R-构型被硫取代的一种新型DNA修饰。目前,磷硫酰化修饰在多种细菌、古生菌以及人类致病菌中多有发现,但其分子调控机制尚不清楚。为了全面解析磷硫酰化修饰的调控机制,本文选择荧光假单胞菌Pf0-1为研究对象,开展了其DNA磷硫酰化修饰的调控机制研究。[方法]首先,构建了spfB基因缺失和回补菌株,使用碘能特异性断裂磷硫酰化修饰DNA的方法,研究了该基因缺失对修饰表型的影响。利用cDNA在相邻同方向的基因间隔区进行PCR,确定了磷硫酰化修饰基因簇spfBCDE内的共转录单元。通过荧光定量RT-PCR,分析了spfB基因缺失突变株中磷硫酰化修饰基因的转录量。利用异源表达并纯化得到的重组蛋白SpfB进行了体外功能研究。通过EMSA实验,验证了SpfB蛋白具有与spfB启动子序列结合活性。通过DNase I footprinting实验,精确定位了SpfB蛋白与DNA结合序列。[结果]spfB基因的缺失加剧了磷硫酰化修饰DNA断裂所致电泳条带弥散的表型,spfB基因的回补能够恢复该表型,证明spfB基因负调控磷硫酰化修饰。鉴定了spf基因簇中只含有1个共转录单元,且该共转录单元在△spfB突变株中转录水平明显上升。通过EMSA和DNase I footprint实验,检测了SpfB蛋白与磷硫酰化修饰基因spfBCDE的启动子区域5''-TGTTTGT-3''相结合。[结论]SpfB作为转录调控因子负调控磷硫酰化修饰基因spfBCDE的表达,为解析磷硫酰化修饰的调控机制和全面理解基因组上的部分修饰特征奠定了基础。     

PNAS：DNA骨架硫修饰研究获新成果

上海交通大学、武汉大学与美国麻省理工学院共同合作联合完成的论文《DNA磷硫酰化修饰在细菌基因组中广泛分布且量化存在》日前在美国《国家科学院院刊》上发表,这是DNA骨架上硫修饰研究领域又一个新的重大进展,也是邓子新团队与彼得·帝丹合作报道了DNA骨架上硫修饰化学本质后持续合作的新成果。     

IV型限制酶ScoMcrA中SRA结构域介导的二聚体化对硫结合结构域功能的影响机制

[背景] 部分细菌的DNA骨架会发生磷硫酰化修饰,硫结合结构域（Sulfur Binding Domain,SBD）可以特异性识别这种生理修饰。与绝大多数SBD-HNH双结构域核酸酶不同,ScoMcrA的SBD和HNH结构域中间插入了一个特异性识别5-甲基胞嘧啶（5mC）修饰DNA的SET and RING-Associated （SRA）结构域。晶体结构显示,单独的SBD是单体,而SBD-SRA是双体。[目的] 探究ScoMcrA中SRA结构域的存在对SBD识别硫修饰DNA的影响及影响方式。[方法] 凝胶迁移实验（Electrophoresis Mobility Shift Assay,EMSA）比较SBD、SBD-SRA对硫修饰DNA结合力的差异;对参与SBD-SRA二聚体化的关键氨基酸残基突变,并检测点突变对SBD-SRA蛋白二聚体化及结合硫修饰DNA的影响。[结果] 相较于SBD结构域,SBD-SRA双结构域对磷硫酰化修饰DNA的结合能力明显增强。对SBD-SRA双体互作界面进行单点突变基本不影响其对硫修饰DNA的结合,当二聚体化界面连续的L₂₆₁LGET₂₆₅突变成A₂₆₁AAAA₂₆₅时,突变体对硫修饰DNA的结合力下降到与SBD相似的水平。[结论] 根据EMSA实验结果可以初步判断,SRA结构域介导的SBD-SRA双体化能增强SBD对硫修饰DNA的结合力;L₂₆₁LGET₂₆₅是SRA结构域上影响SBD对硫修饰DNA结合力的关键氨基酸位点。     

DNA末端硫修饰依赖的体外大片段DNA连接法

【背景】DNA组装技术是基因组合成中的一个关键技术。探索低成本、高效率的基因组合成技术一直是合成生物学的重要研究领域。在某些细菌如变铅青链霉菌中,DNA上有磷硫酰化修饰(简称硫修饰),而在另一些细菌如天蓝色链霉菌中存在一种含有硫修饰识别结构域(sulfur-binding domain, SBD)的识别蛋白,可以特异性识别DNA上的硫修饰,这启发了我们发展出一种新的DNA组装技术。【目的】探究在DNA末端硫修饰的连接中,T4 DNA连接酶与SBD相融合蛋白和单独的T4 DNA连接酶相比,是否有更高的连接效率。【方法】根据同源重组原理,设计硫修饰引物,扩增硫修饰的DNA片段。构建T4 DNA连接酶与SBD融合蛋白的3种表达载体T4-linker-SBD(Hga)、T4-linker-SBD(Spr)和T4-linker-SBD(Mmo),表达纯化以上3种融合蛋白。比较3组浓度梯度(2.4、0.24、0.024 mg/mL) T4 DNA连接酶与融合蛋白在2.5 kb和8.0 kb DNA片段连接上的差异。【结果】DNA末端硫修饰的2.5kb和8.0kb的两端片段均能扩增,而且3种融合蛋白...     

灭癌素链霉菌中磷硫酰化修饰DNA结合结构域的功能分析

[目的]DNA磷硫酰化(phosphorothioation,PT)是由硫原子取代DNA骨架磷原子上的非桥联氧原子形成的一种新型DNA修饰.PT修饰除参与组成限制修饰系统外,其更为广泛的生物学功能仍有待揭示.PT修饰现有的检测方法操作复杂、成本高、耗时长,而具有操作简便、成本低、耗时短等特点的酶联免疫检测(enzyme...     

Ⅳ型限制酶ScoMcrA中SRA结构域介导的二聚体化对硫结合结构域功能的影响机制

【背景】部分细菌的DNA骨架会发生磷硫酰化修饰,硫结合结构域(Sulfur Binding Domain,SBD)可以特异性识别这种生理修饰。与绝大多数SBD-HNH双结构域核酸酶不同,ScoMcrA的SBD和HNH结构域中间插入了一个特异性识别5-甲基胞嘧啶(5m C)修饰DNA的SET and RING-Associated(SRA)结构域。晶体结构显示,单独的SBD是单体,而SBD-SRA是双体。【目的】探究ScoMcrA中SRA结构域的存在对SBD识别硫修饰DNA的影响及影响方式。【方法】凝胶迁移实验(Electrophoresis Mobility Shift Assay,EMSA)比较SBD、SBD-SRA对硫修饰DNA结合力的差异;对参与SBD-SRA二聚体化的关键氨基酸残基突变,并检测点突变对SBD-SRA蛋白二聚体化及结合硫修饰DNA的影响。【结果】相较于SBD结构域,SBD-SRA双结构域对磷硫酰化修饰DNA的结合能力明显增强。对SBD-SRA双体互作界面进行单点突变基本不影响其对硫修饰DNA的结合,当二聚体化界面连续的L261LGET265突变成A261AAAA265时,突变体对硫修饰DNA的结合力下降到与SBD相似的水平。【结论】根据EMSA实验结果可以初步判断,SRA结构域介导的SBD-SRA双体化能增强SBD对硫修饰DNA的结合力;L261LGET265是SRA结构域上影响SBD对硫修饰DNA结合力的关键氨基酸位点。     

沙门氏菌中dnd基因簇的缺失与异源表达

沙门氏菌Salmonella enterica serovar Cerro 87是一株从鸡场分离到的菌株, 其DNA骨架上的磷硫酰化导致了在高压脉冲电泳过程中DNA降解(DNA degradation, Dnd表型)。本研究采用SacB所介导的负筛选系统, 在该菌株中成功缺失了dnd基因簇, 构建了突变株XTG103, 该突变株不再具有Dnd表型。通过异丙基-b-D硫代半乳糖苷(IPTG)诱导启动子PlacZ的转录可以调控DNA磷硫酰化修饰的dnd基因簇的异源表达。     

蛋白质亚磺酰化修饰及其在生理和病理过程中的作用

细胞正常生理或病理过程中均伴随着活性氧(ROS)和活性氮(RNS)的产生，引起蛋白质半胱氨酸发生氧化翻译后修饰。亚磺酰化是氧化翻译后修饰中的一种，指ROS将蛋白质的巯基氧化成亚磺酰基(R-SOH)的过程，广泛存在于多种物种中。亚磺酰化修饰蛋白质的捕获、富集和修饰位点的确定目前仍极具挑战性。半胱氨酸亚磺酰化的检测方法主要包括基于转录因子Yap1和基于小分子化合物dimedone或bicyclo[6.1.0]nonyne的分子探针。在此基础上，研究人员通过偶联生物素等标签分子又设计出了更多便于富集亚磺酰化蛋白质的衍生物探针。将亚磺酰化蛋白质捕获和富集后，与LC-MS/MS等质谱分析技术联用，则可确定发生亚磺酰化修饰的半胱氨酸位点。近几年的研究表明，细胞信号通路中的许多蛋白质或酶都会发生亚磺酰化修饰，调控蛋白质功能、稳定性或催化活性，从而引起下游信号通路或代谢过程的变化，进而影响机体生理或病理状态。随着对蛋白质亚磺酰化修饰的深入研究，越来越多疾病的发生发展新机制被发现，靶向该修饰有望为疾病治疗提供新的策略。本文从蛋白质氧化修饰的过程和亚磺酰化修饰检测的方法入手进行阐述，总结了近几年亚磺酰化修...     

非组蛋白巴豆酰化修饰的生物学功能研究进展

赖氨酸巴豆酰化是一种新近发现的蛋白质翻译后修饰类型,在基因表达、细胞代谢及疾病治疗等许多病理生理过程中都具有重要的调节意义,可能是潜在的药物新靶标。目前研究多关注于组蛋白巴豆酰化修饰,而非组蛋白巴豆酰化修饰的研究逐渐被重视。本文简要介绍非组蛋白巴豆酰化修饰的生物学功能及其在疾病中的作用,将有助于了解非组蛋白巴豆酰化修饰的功能和机制。     

Twenty years hunting for sulfur in DNA

Here we tell a 20-year long story. It began with an easily overlooked DNA degradation (Dnd) phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by five dnd genes. This unusual DNA modification, called phosphorothioation, is the first physiological modification identified on the DNA backbone, in which the nonbridging oxygen is replaced by sulfur in a sequence selective and stereo-specific manner. Homologous dnd gene clusters have been identified in diverse and distantly related bacteria and thus have drawn immediate attention of the entire microbial scientific community. Here, we summarize the progress in chemical, genetic, enzymatic, bioinformatical and analytical aspects of this novel postreplicative DNA modification. We also discuss perspectives on the physiological functions of the DNA phosphorothioate modification in bacteria and their implications.     

Triplex formation of chemically modified homopyrimidine oligonucleotides: thermodynamic and kinetic studies.

We have investigated effects of chemical modifications of a third strand on the thermodynamic and kinetic properties of the triplex formation between a 23-bp duplex and each of four kinds of 15-mer chemically modified third strands using isothermal titration calorimetry and interaction analysis system. The chemical modifications of the third strand included one base modification, with replacement of thymine by uracil; two sugar moiety modifications, RNA and 2'-O-methyl-RNA; and one phosphate backbone modification, with replacement of phosphodiester by phosphorothioate backbone. The thermodynamic and kinetic parameters obtained were similar in magnitude at room temperature for the triplex formation with the base-modified and the sugar-modified third strands. By contrast, binding constant for the triplex formation with the third strand containing phosphorothioate backbone was much smaller by a factor of 10 than that for the other triplex formations. Kinetic analyses have also demonstrated that the third strand containing phosphorothioate backbone was much slower in the association step and much faster in the dissociation step than the other third strands, which resulted in the much smaller binding constant. The reason for the instability of the triplex with the third strand containing phosphorothioate backbone will be discussed. We conclude that, at least in the triplex formation with the chemically modified third strands studied in the present work, the modification of phosphate backbone of the third strand produces more significant effect on the triplex formation than the modifications of base and sugar moiety.     

In Vivo Mutational Characterization of DndE Involved in DNA Phosphorothioate Modification

DNA phosphorothioate (PT) modification is a recently identified epigenetic modification that occurs in the sugar-phosphate backbone of prokaryotic DNA. Previous studies have demonstrated that DNA PT modification is governed by the five DndABCDE proteins in a sequence-selective and R _P stereo-specific manner. Bacteria may have acquired this physiological modification along with dndFGH as a restriction-modification system. However, little is known about the biological function of Dnd proteins, especially the smallest protein, DndE, in the PT modification pathway. DndE was reported to be a DNA-binding protein with a preference for nicked dsDNA in vitro; the binding of DndE to DNA occurs via six positively charged lysine residues on its surface. The substitution of these key lysine residues significantly decreased the DNA binding affinities of DndE proteins to undetectable levels. In this study, we conducted site-directed mutagenesis of dndE on a plasmid and measured DNA PT modifications under physiological conditions by mass spectrometry. We observed distinctive differences from the in vitro binding assays. Several mutants with lysine residues mutated to alanine decreased the total frequency of PT modifications, but none of the mutants completely eliminated PT modification. Our results suggest that the nicked dsDNA-binding capacity of DndE may not be crucial for PT modification and/or that DndE may have other biological functions in addition to binding to dsDNA.     

化学修饰对反义寡核苷酸稳定性及抗流感病毒活性的影响

为了探讨 Ａ Ｓ Ｏ Ｄ Ｎ 化学修饰形式与 Ａ Ｓ Ｏ Ｄ Ｎ 稳定性,体外细胞毒性以及抗流感病毒活性之间的关系,合成了 ７ 种不同化学修饰形式的 Ａ Ｓ Ｏ Ｄ Ｎ：硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰;３′与 ５′端硫代,中间为天然结构的混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端分别磷酸化和胆固醇修饰等．测定了 ７ 种修饰体在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液,含 ２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ培养液以及水中的稳定性,体外细胞毒性和在细胞水平抗流感病毒活性．结果表明,混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ,硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇的修饰形式在小鼠血清, Ｍ Ｄ Ｃ Ｋ 细胞裂解液与含２％ 胎牛血清的 Ｄ Ｍ Ｅ Ｍ 培养液中稳定性相对较高,作用 ２４～４８ ｈ 仅混合骨架 Ａ Ｓ Ｏ Ｄ Ｎ 与硫代 Ａ Ｓ Ｏ Ｄ Ｎ 发生部分降解;天然结构 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆固醇修饰体在 ２４ ｈ 内大部分降解．所有 Ａ Ｓ Ｏ Ｄ Ｎ 修饰体在水中具有很高稳定性,４８ ｈ 内未见降解作用．７ 种 Ａ Ｓ Ｏ Ｄ Ｎ 修饰形式在 Ｍ Ｄ Ｃ Ｋ 细胞中未表现明显的细胞毒性．硫代 Ａ Ｓ Ｏ Ｄ Ｎ 及其 ３′端接磷酸和胆     

Reactive derivatives of oligonucleotide phosphorothioate analogues: IV. Site-directed modification of nucleic acids and intramolecular alkylation of phosphorothioate groups

Alkylation of the 22-mer DNA target pTGCCTGGAGCTGCTTGATGCCC (I) by oligodeoxynucleotide phosphorothioate derivatives (PTAO) GpsCpsApsTpsCpsApsApsGpsCpsApsGpsCpN(CH₃)CH₂(RCl)(II-PS) and (RCl)CH₂N(CH₃)pGpsCpsAps TpsCpsApsApsGpsCpsApsGpsC (III-PS) bearing a residue of an aromatic analogue of nitrogen lost (RCl=C₆H₄N(CH₃)(CH₂CH₂Cl) at the 3′- or 5′-end was studied. It was shown that the internucleotide phosphorothioate bonds do not affect the regiospecificity of the target modification. The maximum degree of the target modification (att→∞) at 20°C was about 25% for both (II-PS) and (III-PS). The use of GCATCAAGCAGCpN(CH₃)CH₂(RCl)(II-PO), containing internucleotide phosphodiester bonds, under the same conditions gave about 65% of the modified DNA. Kinetics of the PTAO-induced complementarily addressed nucleic acid (NA) modification was analyzed. The rate constants of the reaction of the intermediate reactive ethylenimmonium ion with phosphorothioate groups of the reagents were evaluated both in solution and in duplex. The intramolecular alkylation of phosphorothioate groups considerably affected the DNA target modification by decreasing the effectiveness of the modification in a wide range of temperatures and changing the temperature dependence of the modification from a bell-like to an S-like profile. It was concluded that, in the course of the modification, the PTAO phosphorothioate groups are intramolecularly alkylated both in solution and in the complementary NA target-oligonucleotide duplex. For Part III, see [1].     

Evidence for sequence-specific conformational changes in DNA from the melting temperatures of DNA phosphorothioate derivatives.

Analogs of alternating purine-pyrimidine DNA polymers such as poly(dA-dT)-poly(dA-dT) can be made with phosphorothioate groups in the DNA backbone. A phosphorothioate diester at the 5'-purine-pyrimidine-3' step causes a significant lowering of the polymer's melting temperature compared to a phosphorothioate diester at the 5'-pyrimidine-purine-3' step. This may occur because sulfur substitution increases anionic charge density in the DNA minor groove and 5'-purine-pyrimidine-3' steps narrow the minor groove. The ability to modulate charge density in the DNA backbone via sulfur substitution should prove useful in studies of sequence-dependent conformational changes in DNA.     

Chemical modification of siRNAs to improve serum stability without loss of efficacy

Development of RNA interference as a novel class of therapeutics requires improved pharmacokinetic properties of short interfering RNA (siRNA). To confer enhanced serum stability to Sur10058, a hyperfunctional siRNA which targets survivin mRNA, a systematic modification at the 2'-sugar position and phosphodiester linkage was introduced into Sur10058. End modification of three terminal nucleotides by 2'-OMe and phosphorothioate substitutions resulted in a modest increase in serum stability, with 3' end modification being more effective. Alternating modification by 2'-OMe substitution significantly stabilized Sur10058, whereas phosphorothioate modification was only marginally effective. Through various combinations of 2'-OMe, 2'-F and phosphorothioate modifications that were directed mainly at pyrimidine nucleotides, we have identified several remarkably stable as well as efficient forms of Sur10058. Thus, our results provide an effective means to stabilize siRNA in human serum without compromising the knockdown efficiency. This advancement will prove useful for augmenting the in vivo potency of RNA interference.     

Identification of DNA sequences flanking T-DNA insertions by PCR-walking

In recent years, concerns over genetic modification issues have resulted in regulatory authorities requiring comprehensive analysis of transgene insertion events in the plants that are to be commercialized. Determining that plants are devoid of vector backbone sequences is a trivial task that is best achieved by Southern blot analysis; however, identifying the DNA sequences flanking the T-DNA insertions can be arduous. In this paper, we present a robust method of characterizing this insertion event. We have applied and modified a genomic walking method that combines vectorette and suppression PCR walking.     

A procedure for selective DNA alkylation and detection by mass spectrometry.

A method which improves the detectability of DNA by mass spectrometry is presented. By quantitatively alkylating the backbone of phosphorothioate oligonucleotides the problems of gas phase ion generation by matrix assisted laser desorption ionization can be controlled. We have developed a selective alkylating protocol for phosphorothioate oligonucleotides which is a facile way of generating non-ionic nucleic acids. A variety of alkylating agents was studied and their kinetics were monitored in a gel electrophoretic assay and by mass spectrometry.