DNA脱碱基位点的检测方法及其生物学研究进展

DNA中糖苷键断裂形成的脱碱基位点（脱嘌呤/嘧啶位点,AP位点）是常见的DNA损伤类型之一,由核苷酸自发水解产生,也是DNA碱基切除修复途径中的关键中间体。若修复不及时,可能会导致DNA复制阻滞和DNA链断裂,产生突变和细胞毒性,同时还会引起形成DNA交联或DNA-蛋白质交联的损伤,因此对于AP损伤的检测有助于理解细胞氧化应激损伤和基因毒性物质的毒性评价。目前检测AP位点的方法有14C或32P后标记法、酶联免疫吸附分析（ELISA）法和液相色谱-质谱（LC-MS）技术等。该文重点概述DNA中AP位点的检测方法和生物学研究进展,并展望了AP位点损伤相关研究的前景。     

DNA脱碱基位点的检测与应用

脱碱基位点是一种常见的DNA损伤,源于N-糖苷键断裂而使碱基脱落。辐射、烷基化试剂和一些抗癌药物等可能会造成碱基脱落,因此脱碱基位点作为标志性损伤能够帮助疾病早期筛查、药物毒副作用评价、环境污染物毒性评价等。目前已有不同的检测方法用于脱碱基位点的定量、定性分析,包括32P后标记法、LC-MS、ELISA及化学探针检测法等。另一方面,由于脱碱基位点在双链DNA内形成疏水空腔,能够结合小分子,使得脱碱基位点作为结合位点被用于小分子检测、构建适配体传感器及SNP检测。本文简要概述目前为止对DNA脱碱基位点的化学探针检测法研究进展以及含有脱碱基位点DNA的应用研究进展,并展望其发展趋势。     

序列特异性DNA断裂蛋白质

序列特异性DNA断裂蛋白质是在序列特异性DNA结合蛋白质及小分子DNA断裂试剂基础上设计合成的。其基本原理是在含有DN A结合域的蛋白质上引入一个金属鳌合剂并鳌合一个适当的金属离子,其中序列特异性DNA结合蛋白质具有与DNA特定序列结合的能力,从而可以起导向物的作用,而引入的金属鳌合齐J与金属离子复合物具有断裂DNA的功能,两者协同作用可以达到序列特异性断裂DNA的目的。     

铜配合物促进的DNA氧化断裂

本文首先对铜配合物催化DNA氧化断裂的机制及相关因素包括DNA结合能力与方式、活性氧物种形成和活性氧物种对底物的损伤展开了讨论;其次结合我们课题组的工作对配合物的结构对其DNA切割性能的影响分别进行了总结,分别探讨了核数、核的种类、位阻、电荷、结合方式以及氧化还原电位等因素的影响;同时还对光激发铜配合物断裂DNA的机制和性能进行分析;最后对特异性含铜DNA断裂试剂的不同设计策略及其相关切割行为进行的系统地总结. 这些总结不仅有利于对铜配合物促进DNA 断裂行为的系统理解,而且对于进一步设计高效含铜人工核酸酶实现DNA的特异性断裂具有良好的指导意义.     

氯乙基亚硝基脲烷化碱基导致DNA单链断裂的机理研究

采用密度泛函理论(DFT)和MΦller-Pleset微扰理论(MP)方法对氯乙基亚硝基脲(CENUs)烷化DNA碱基导致单链断裂的机理进行了研究. 对包括CENUs的分解、DNA碱基的烷化、糖苷键的水解、脱嘌呤位点的开环以及最终导致单链断裂的磷酸二酯的消除在内的多步反应过程进行了探讨. 在B3LYP/6-31++G**水平上对各驻点(反应物、中间体、过渡态和生成物)进行了全几何结构优化; 为了模拟细胞环境, 采用自洽场连续极化模型(CPCM)在相同计算水平上对各驻点进行了水相中的单点能计算或全几何结构优化. 分别在B3LYP/6-31++G**和MP2/6-311++G**水平上绘出反应势能曲线, 结果显示, 在整个反应过程中, 磷酸二酯的消除反应能垒最高, 而氯乙基重氮盐离子烷化DNA碱基的反应能垒最低. 理论分析结果表明, CENUs 一旦分解便很容易烷化DNA碱基, 随后生成的氯乙基化鸟嘌呤会迅速从DNA链上脱去, 尽管如此, 脱嘌呤位点最终导致DNA单链断裂的一系列反应则是一个缓慢的过程, 这与断链反应的动力学实验结果相符合.     

金属离子与DNA相互作用的研究进展

对金属离子与DNA相互作用的研究进展进行了综述,讨论了不同金属离子与DNA碱基的作用位点以及不同金属离子与DNA相互作用的特点.     

DNA脱碱基位点存在下非氢键配体的增强结合研究

众所周知,插入剂的DNA特性结合位点位于DNA碱基对之间,然而这种非共价相互作用对于含脱碱基（AP）位点的DNA来讲还没有引起足够的重视,虽然在生物细胞中总是存在着DNA脱碱基位点。本文以原黄素（proflavine,PF）为例研究了插入剂对DNA中AP位点的结合特性。实验结果表明,相对于插入位点而言,AP位点是PF的优先结合位点,AP位点的本征结合常数比插入结合常数高一个数量级以上。此外,PF的结合使含脱碱基位点DNA的热稳定性明显提高,表明PF在脱碱基位点的结合构像明显不同于插入结合时的分子定向。本文结果将有助于判断小分子的DNA结合方式所决定的药物的生物化学及生物物理效用。     

具有强烈杀伤肿瘤细胞活性的新抗肿瘤抗生素C1027分子作用机制

本研究表明,C1027是以细胞内染色质或染色体DNA为靶体,直接造成核小体连接区DNA损伤,损伤的方式包括DNA单链断裂、双链断裂及单链断裂合并互补链上相邻断裂口部位出现无碱基位点,C1027损伤细胞DNA的作用显著强于新制癌菌素,平阳霉素,阿霉素及丝裂霉素C,本文还从分子机制的水平讨论了C1027强烈细胞毒性。     

二价金属离子激活铜超氧化物歧化酶断裂DNA的活性

以铜超氧化物歧化酶(CunSOD, n＝1—4)作为野生型CuZnSOD突变体的一种模型, 研究了其在二价金属离子(Mg2+, Mn2+)存在下由非氧化途径断裂DNA的活性, 并与CuZnSOD和脱辅基SOD(apoSOD)进行了比较. 结果表明, 在Mg2+或Mn2+存在下, CunSOD断裂DNA的活性高于CuZnSOD和apoSOD, 并且DNA的断裂活性受二价金属离子浓度、pH及酶浓度等因素影响. 相对活性及动力学参数的测定结果表明, CunSOD断裂DNA的相对能力按Cu1SOD2SOD≈Cu4SOD3SOD的顺序变化.     

三聚氰胺对DNA潜在损伤作用的研究

在生理酸度条件下(pH 7.4),采用溴化乙锭(EB)为荧光探针的荧光光谱法、I-离子荧光猝灭效应、DNA熔点和粘度效应等手段,研究了三聚氰胺与DNA的相互作用。随着DNA的加入,三聚氰胺的荧光强度明显减小而且三聚氰胺能够猝灭DNA-EB复合物的荧光,说明三聚氰胺能够竞争置换EB而与DNA作用;三聚氰胺的加入使得DNA的粘度增大,DNA-EB的熔点降低;DNA的加入减小了I-对三聚氰胺荧光的猝灭程度。三聚氰胺以嵌插方式作用于DNA的亲核位点,意味着三聚氰胺进入生物体后有可能通过形成DNA加合物的形式造成DNA损伤,从而最终导致基因突变。     

Electrochemical DNA Hybridization Detection Using DNA Cleavage

We report the new method for detection of DNA hybridization using enzymatic cleavage. The strategy is based on that S1 nuclease is able to specifically cleave only single strand DNA, but not double strand DNA. The capture probe DNA, thiolated single strand DNA labeled with electroactive ferrocene group, was immobilized on a gold electrode. After hybridization of target DNA of complementary and noncomplementary sequences, nonhybridized single strand DNA was cleaved using S1 nuclease. The difference of enzymatic cleavage on the modified gold electrode was characterized by cyclic voltammetry and differential pulse voltammetry. We successfully applied this method to the sequence‐selective discrimination between perfectly matched and mismatched target DNA including a single‐base mismatched target DNA. Our method does not require either hybridization indicators or other exogenous signaling molecules which most of the electrochemical hybridization detection systems require.     

Metallodesferals as a new class of DNA cleavers: Specificity, mechanism and targetting of DNA scission reactions

Interaction of metal complexes with nucleic acids is currently attracting wide attention due to their potential utility as drugs, regulators of gene expression and tools for molecular biology. Many metal complexes exhibit nucleolytic activity, the most important examples being Cu(II)-OP, Fe(H)-BLM, Fe(II)-EDTA, metalloporphyrins, Ru and Co complexes of 4,7-diphenyl-l,10-phenanthroline and more recently by Ni(II) complexes. Desferal, a well known siderophore and a highly effective drug in chelation therapy of iron overload diseases, forms a stable octahedral co-ordination Fe(III) complex Eerrioxamine B. We have been interested in the DNA damaging properties of metallodesferals and this paper describes the DNA cleaving ability of metallodesferals, metal-dependent base selectively in DNA scission reactions, mechanistic studies on DNA cleavage by CuDFO and targetting of DNA cutting by covalent MDFO conjugates. This paper reports the synthesis of Cu(II), Co (III) and Ni(II) complexes of a siderophore chelating drug desferal, the studies on cleavage of plasmid DNA, the sequence preference of cleavage reactions, and C1’ as the primary site of hydroxyl radical attack in the reactions. Oligonucleotides covalently linked with this molecular scissor can direct the cleavage of either single or double strand DNA’s, mediated by duplex or triple helix structures respectively. Such targetting of DNA cleavage reactions, mediated by oligonucleotide-Cu(II)/Co(III) desferal conjugates has demonstrated reasonable site specificity and efficiency     

Use of an automated capillary DNA sequencer to investigate the interaction of cisplatin with telomeric DNA sequences

The determination of the sequence selectivity of DNA-damaging agents is very important in elucidating the mechanism of action of anti-tumour drugs. The development of automated capillary DNA sequencers with fluorescent labelling has enabled a more precise method for DNA sequence specificity analysis. In this work we utilized the ABI 3730 capillary sequencer with laser-induced fluorescence to examine the sequence selectivity of cisplatin with purified DNA sequences. The use of this automated machine enabled a higher degree of precision of both position and intensity of cisplatin-DNA adducts than previously possible with manual and automated slab gel procedures. A problem with artefact bands was overcome by ethanol precipitation. It was found that cisplatin strongly formed adducts with telomeric DNA sequences.     

Carbon—Hydrogen Bonds of DNA Sugar Units as Targets for Chemical Nucleases and Drugs

This review article focuses on the molecular aspects of DNA cleavage by synthetic chemical nucleases (transition metal complexes endowed with redox properties and DNA affinity) and natural drugs (cytotoxic agents such as bleomycins or enediynes). Unlike deoxyribonucleases, which catalyze the nucleophilic attack of water on the phosphorus atom of a particular phosphodiester entity, these nonhydrolytic DNA-cleavers are able to oxidize the sugar units, generally by hydrogen atom abstraction. Examples of oxidative attack on each of the five different C? H bonds of deoxyribose are known, depending on the nature, structure, type of activation, or mode of DNA interaction of the DNA-cleaver. Further evolution at the site of the initial lesion leads to the release of bases, oxidized deoxyribose units, or oxidized sugar fragments appended to the base or the terminal phosphate. In most cases the loss of a part (at least) of a nucleoside, with the concomitant loss of one base information, primarily induces the cleavage of the DNA strand. For both types of DNA cleavage reagents studied within the two last decades, the modes of activation and DNA binding are presented, as well as the details on the mechanism of deoxyribose oxidative degration. Because of the need for highly efficient and highly specific reagents, the development of new artificial and selective DNA cleavers, supported by an improved knowledge of these different mechanisms of DNA cleavage, is to-day a challenging area in the rational design of antitumoral or antiviral agents, as well as in the field of molecular biology.     

Reductive electron transfer in phenothiazine-modified DNA is dependent on the base sequence

A new DNA assay has been designed, prepared and applied for the chemical investigation of reductive electron transfer through the DNA. It consists of 5-(10-methyl-phenothiazin-3-yl)-2'-deoxyuridine (Ptz-dU, 1) as the photoexcitable electron injector and 5-bromo-2'-deoxyuridine (Br-dU) as the electron trap. The Ptz-dU-modified oligonucleotides were synthesised by means of a Suzuki-Miyaura cross-coupling protocol and subsequent automated phosphoramidite chemistry. Br-dU represents a kinetic electron trap, since it undergoes a chemical modification after its one-electron reduction that can be analysed by piperidine-induced strand cleavage. The quantification of the strand cleavage yields from irradiation experiments reveals important information about the electron-transfer efficiency. The performed DNA studies focused on the base sequence dependence of the electron-transfer efficiency with respect to the proposal that C*- and T*- act as intermediate electron carriers during electron hopping. From our observations it became evident that excess-electron transfer is highly sequence dependent and occurs more efficiently over T-A base pairs than over C-G base pairs.     

Rational engineering of a DNA glycosylase specific for an unnatural cytosine:pyrene base pair

A novel site-specific cytosine DNA glycosylase has been rationally engineered from the active site scaffold of the DNA repair enzyme uracil DNA glycosylase (UDG). UDG, which operates by a nucleotide flipping mechanism, was first converted into a sequence nonspecific cytosine DNA glycosylase (CDG) by altering the base-specific hydrogen bond donor-acceptor groups in the active site. A second mutation that renders UDG defective in nucleotide flipping was then introduced, and the double mutant was rescued using a substrate with a "preflipped" cytosine base. Substrate-assisted flipping was engineered by incorporation of an unnatural pyrene nucleotide wedge (Y) into the DNA strand opposite to the target cytosine. This new enzyme, CYDG, can be used to target cleavage of specific cytosine residues in the context of a C/Y base pair in any DNA fragment.     

Electron Transfer through DNA in the Course of Radical-Induced Strand Cleavage

No benefit from base stacking is observed for rates of electron transfer in DNA. This conclusion was drawn from experiments with a new DNA assay in which a radical cationic site, generated by strand cleavage, can be reduced by the guanine bases in the same DNA (the electron transfer is indicated by arrows in the diagram). The distance dependence of this electron transfer step is determined by the chemical yield of the reduction product.     

One-electron photooxidation and site-selective strand cleavage at 5-methylcytosine in DNA by sensitization with 2-methyl-1,4-naphthoquinone-tethered oligonucleotides

Photosensitized one-electron oxidation was applied to discriminate a specific base site of 5-methylcytosine (mC) generated in DNA possessing a partial sequence of naturally occurring p53 gene, using a sensitizing 2-methyl-1,4-naphthoquinone (NQ) chromophore tethered to an interior of oligodeoxynucleotide (ODN) strands. Photoirradiation and subsequent hot piperidine treatment of the duplex consisting of mC-containing DNA and NQ-tethered complementary ODN led to oxidative strand cleavage selectively at the mC site, when the NQ chromophore was arranged so as to be in close contact with the target mC. The target mC is most likely to be one-electron oxidized into the radical cation intermediate by the sensitization of NQ. The resulting mC radical cation may undergo rapid deprotonation and subsequent addition of molecular oxygen, thereby leading to its degradation followed by strand cleavage at the target mC site. In contrast to mC-containing ODN, ODN analogs with replacement of normal cytosine, thymine, adenine, or guanine at the mC site underwent less amount of such an oxidative strand cleavage at the target base site, presumably due to occurrence of charge transfer and charge recombination processes between the base radical cation and the NQ radical anion. Furthermore, well designed incorporation of the NQ chromophore into an interior of ODN could suppress a competitive strand cleavage at consecutive guanines, which occurred as a result of positive charge transfer. Thus, photosensitization by an NQ-tethered ODN led to one-electron oxidative strand cleavage exclusively at the target mC site, providing a convenient method of discriminating mC in naturally occurring DNA such as human p53 gene as a positive band on a sequencing gel.