Study of the Mode and Efficiency of DNA Binding in the Damage Induced by Photoactivated Water Soluble Porphyrins

We have investigated the DNA binding interactions and in vitro photoactivated DNA damage induced by a neutral water soluble porphyrin derivative 5,10,15,20‐tetrakis(2,4,6‐trihydroxyphenyl)porphyrin (TTHPP) and its zinc derivative 5,10,15,20‐tetrakis(2,4,6‐trihydroxyphenyl)porphyrinato zinc(II) (Zn‐TTHPP) upon visible light irradiation through various spectroscopic techniques and employing repair endonucleases. These porphyrin derivatives exhibited high affinity toward DNA through groove binding interactions as evidenced through the UV–vis absorption, emission, circular dichroism spectral and viscosity changes. Interestingly, the free base porphyrin derivative, TTHPP generated efficient singlet oxygen mediated DNA damage sensitive to formamidopyrimidine‐DNA glycosylase (Fpg protein), when compared with its metal derivative and to the well‐known photosensitizer, hematoporphyrin. These results provide direct evidence for the role of DNA binding mode as well as extent of interactions with DNA in the efficiency of photoactivated DNA damage induced by the neutral porphyrins, which are believed to be the ideal candidates for photodynamic therapeutic applications.     

Controlling protein activity with ligand-regulated RNA aptamers

Controlling the activity of a protein is necessary for defining its function in vivo. RNA aptamers are capable of inhibiting proteins with high affinity and specificity, but this effect is not readily reversible. We describe a general method for discovering aptamers that bind and inhibit their target protein, but addition of a specific small molecule disrupts the protein-RNA complex. A SELEX protocol was used to raise RNA aptamers to the DNA repair enzyme, formamidopyrimidine glycosylase (Fpg), and neomycin was employed in each round to dissociate Fpg-bound RNAs. We identified an RNA molecule able to completely inhibit Fpg at 100 nM concentration. Importantly, Fpg activity is recovered by the addition of neomycin. We envision these ligand-regulated aptamers (LIRAs) as valuable tools in the study of biological phenomena in which the timing of molecular events is critical.     

Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli

8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.     

SPR imaging for label-free multiplexed analyses of DNA N-glycosylase interactions with damaged DNA duplexes

Base excision repair (BER) is the major mechanism for the correction of damaged nucleobases resulting from the alkylation and oxidation of DNA. The first step in the BER pathway consists of excision of the abnormal base by several specific DNA N-glycosylases. A decrease in BER activity was found to be related to an increased risk of carcinogenesis and aging. To investigate BER activities we set up a new device for DNA repair analysis based on surface plasmon resonance imaging (SPRi). Oligonucleotides bearing an abnormal nucleoside, namely 8-oxo-7,8-dihydro-2'-deoxyguanosine and (5'S)-5',8-cyclopurine-2'-deoxynucleoside, were grafted by a pyrrole electro-copolymerization process on a glass prism coated with a gold layer. The latter label-free DNA sensor chip permits the detection of N-glycosylase/AP-lyase activity as well as the binding of repair proteins to DNA damage without cleavage activity. Thus, the Fapy DNA N-glycosylase (Fpg) protein is shown as expected to bind and then cleave its natural substrate, namely 8-oxo-7,8-dihydro-guanine, together with the resulting abasic site. Using the current SPR imaging-based DNA array we observed an original binding activity of Fpg towards the (5'S)-5',8-cyclodAdenosine residue. These results altogether show that SPR imaging may be used to simultaneously and specifically detect recognition and excision of several damaged DNA nucleobases, and constitutes an interesting technique to screen inhibitors of DNA repair proteins.     

Molecular simulations reveal a common binding mode for glycosylase binding of oxidatively damaged DNA lesions

Cellular DNA is constantly exposed to oxidative stress from both exogenous and endogenous sources, creating lesions that lead to aging related diseases, including cancer. 8-Oxo-guanine (8OG) is one of the most common forms of oxidative DNA damage, and failure to repair this lesion results in G:C to T:A transversion. Another common lesion, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapydG), shares the same precursor as 8OG. In Escherichia coli, both lesions are recognized and excised by the DNA glycosylase Fpg. X-ray crystallographic studies have shown that FapydG and 8OG adopt different conformations in the active site of Fpg. Our simulations suggest that the different binding modes observed for 8OG and FapydG arise directly from response to the nonconserved E77 present in the thermophilic Fpg sequences used for the crystallography experiments. In simulations with consensus S77, these lesions adopt very similar binding modes.     

Molecular mechanics parameters for the FapydG DNA lesion

FapydG is a common oxidative DNA lesion involving opening of the imidazole ring. It shares the same precursor as 8-oxodG and can be excised by the same enzymes as 8-oxodG. However, the loss of the aromatic imidazole in FapydG results in a reduction of the double bond character between C5 and N7, with an accompanying increase in conformational flexibility. Experimental characterization of FapydG is hampered by high reactivity, and thus it is desirable to investigate structural details through computer simulation. We show that the existing Amber force field parameters for FapydG do not reproduce X-ray structural data. We employed quantum mechanics energy profile calculations to derive new molecular mechanics parameters for the rotation of the dihedral angles in the eximidazole moiety. Using these parameters, all-atom simulations in explicit water reproduce the nonplanar conformation of cFapydG in the crystal structure of the complex with L. lactis glycosylase Fpg. We note that the nonplanar structure is stabilized by an acidic residue that is not present in most Fpg sequences. Simulations of the E-->S mutant, as present in E. coli, resulted in a more planar conformation, suggesting that the highly nonplanar form observed in the crystal structure may not have direct biological relevance for FapydG.     

Surprising repair activities of nonpolar analogs of 8-oxoG expose features of recognition and catalysis by base excision repair glycosylases

Repair glycosylases locate and excise damaged bases from DNA, playing central roles in preservation of the genome and prevention of disease. Two key glycosylases, Fpg and hOGG1, function to remove the mutagenic oxidized base 8-oxoG (OG) from DNA. To investigate the relative contributions of conformational preferences, leaving group ability, enzyme-base hydrogen bonding, and nucleobase shape on damage recognition by these glycosylases, a series of four substituted indole nucleosides, based on the parent OG nonpolar isostere 2Cl-4F-indole, were tested as possible direct substrates of these enzymes in the context of 30 base pair duplexes paired with C. Surprisingly, single-turnover experiments revealed that Fpg-catalyzed base removal activity of two of the nonpolar analogs was superior to the native OG substrate. The hOGG1 glycosylase was also found to catalyze removal of three of the nonpolar analogs, albeit considerably less efficiently than removal of OG. Of note, the analog that was completely resistant to hOGG1-catalyzed excision has a chloro-substituent at the position of NH7 of OG, implicating the importance of recognition of this position in catalysis. Both hOGG1 and Fpg retained high affinity for the duplexes containing the nonpolar isosteres. These studies show that hydrogen bonds between base and enzyme are not needed for efficient damage recognition and repair by Fpg and underscore the importance of facile extrusion from the helix in its damaged base selection. In contrast, damage removal by hOGG1 is sensitive to both hydrogen bonding groups and nucleobase shape. The relative rates of excision of the analogs with the two glycosylases highlight key differences in their mechanisms of damaged base recognition and removal.     

Identification of Escherichia coli 8-oxoguanine endonuclease

7,8-Dihydro-8-oxoguanine (oh8Gua) endonuclease is a DNA repair enzyme in Escherichia coli to remove oh8Gua, a promutagenic DNA adduct. Due to the unique mode of enzyme action and substrate specificity, this DNA repair enzyme has been suggested to be identical to 2,6-diamino-4-hydroxyformamidopyrimidine (Fapy)-DNA glycosylase (Fpg). However, oh8Gua endonuclease had not been definitely identified because it had not been homogeneously purified. In this study, we attempted to purify and identify the enzyme. Through several purification procedures, we obtained two proteins (32 kD and 29 kD). The larger protein co-migrated with Fpg in 12% SDS-PAGE gel. Sequences of N-terminal amino acids of these two proteins were identical to that of Fpg; the smaller one is a degraded product of oh8Gua endonuclease during purification steps. These results indicate that oh8Gua endonuclease is identical to Fpg, implying that oh8Gua in oxidatively damaged DNA rather than Fapy is more physiologically relevant substrate for Fpg.     

DNA quality control by conformational readout on the undamaged strand of the double helix

Synthetic DNA probes were incubated in human cell extracts to dissect the early step of bulky lesion recognition in the nucleotide excision repair pathway. Excision was induced upon combination of the target adduct with either a two-sided bulge, involving both the damaged sequence and its undamaged partner strand, or a one-sided bulge, affecting exclusively the undamaged complementary sequence. Surprisingly, the same adduct became refractory to repair when only the modified strand was bulged out of the double helix. Adduct removal was further dependent on an intact opposing strand and, at carcinogen-DNA adducts, the assembly of excision complexes was triggered by a single flipped-out deoxyribonucleotide in the complementary sequence. These findings describe a mechanism of molecular readout in DNA repair that, unexpectedly, is entirely confined to the undamaged side of the double helix.     

Fluorescence polarization detection for affinity capillary electrophoresis

Affinity capillary electrophoresis (ACE) with laser-induced fluorescence polarization (LIFP) detection is described, with examples of affinity interaction studies. Because fluorescence polarization is sensitive to changes in the rotational motion arising from molecular association or dissociation, ACE-LIFP is capable of providing information on the formation of affinity complexes prior to or during CE separation. Unbound, small fluorescent probes generally have little fluorescence polarization because of rapid rotation of the molecule in solution. When the small fluorescent probe is bound to a larger affinity agent, such as an antibody, the fluorescence polarization (and anisotropy) increases due to slower motion of the much larger complex molecule in the solution. Fluorescence polarization results are obtained by simultaneously measuring fluorescence intensities of vertical and horizontal polarization planes. Applications of CE-LIFP to both strong and weak binding systems are discussed with antibody-antigen and DNA-protein binding as examples. For strong affinity binding, such as between cyclosporine and its antibody, complexes are formed prior to CE-LIFP analysis. For weaker binding, such as between single-stranded DNA and its binding protein, the single-stranded DNA binding protein is added to the CE separation buffer to enhance dynamic formation of affinity complexes. Both fluorescence polarization (and anisotropy) and mobility shift results are complementary and are useful for immunoassays and binding studies.     

Probing the requirements for recognition and catalysis in Fpg and MutY with nonpolar adenine isosteres

The Escherichia coli DNA repair enzymes Fpg and MutY are involved in the prevention of mutations resulting from 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) in DNA. The nonpolar isosteres of 2'-deoxyadenosine, 4-methylbenzimidazole beta-deoxynucleoside (B), and 9-methyl-1H-imidazo[4,5-b]pyridine beta-deoxynucleoside (Q), were used to examine the importance of hydrogen bonding within the context of DNA repair. Specifically, the rate of base removal under single-turnover conditions by the MutY and Fpg glycosylases from duplexes containing OG:B and OG:Q mismatches, relative to OG:A mismatches, was evalulated. The reaction of Fpg revealed a 5- and 10-fold increase in rate of removal of OG from duplexes containing OG:B and OG:Q base pairs, respectively, relative to an OG:A mispair. These results suggest that the lack of the ability to hydrogen bond to the opposite base facilitates removal of OG. In contrast, adenine removal catalyzed by MutY was much more efficient from an OG:A mispair-containing duplex (k2 = 12 +/- 2 min(-1)) compared to the removal of B from an OG:B duplex (k(obs) < 0.002 min(-1)). Surprisingly, MutY was able to catalyze base removal from the OG:Q-containing substrate (k2 = 1.2 +/- 0.2 min(-1)). Importantly, the B and Q analogues are not deleterious to high-affinity DNA binding by MutY. In addition, the B and Q analogues are more susceptible to acid-catalyzed depurination illustrating that the enzyme-catalyzed mechanism is distinct from the nonenzymatic mechanism. Taken together, these results point to the importance of both N7 and N3 in the mechanism of adenine excision catalyzed by MutY.     

配体形状对多吡啶铜(Ⅱ)配合物与DNA作用的影响

合成了一系列含有平面配体的Cu(Ⅱ)多吡啶配合物[Cu(IP)₂]²⁺、[Cu(PIP)₂]²⁺、[Cu(DPPZ)₂]²⁺和[Cu(HPIP)₂]²⁺，用吸收光谱、CD光谱和粘度等方法研究了这些配合物与小牛胸腺DNA的作用。结果表明配体上的取代基及配体的平面性对这些四面体配合物与DNA的结合强弱产生一定的影响。[Cu(DPPZ)₂]²⁺与DNA的结合较强，而[Cu(HPIP)₂]²⁺与DNA的结合较弱。CD光谱显示配合物[Cu(DPPZ)₂]²⁺、[Cu(PIP)₂]²⁺和[Cu(HPIP)₂]²⁺的加入会导致DNA的CD光谱减弱。而[Cu(IP)₂]²⁺的加入则会使DNA的CD光谱增强。同时，[Cu(IP)₂]²⁺与DNA结合后，还会引起一定程度的DNA构型转换，即DNA从B型转换成Z型。     

Synthesis and spectroscopic DNA binding studies of [Ru(phen)2(tbtc)]2+ and [Ru(2,9-dmp)2(tbtc)]2+

A new polypyridyl ligand tbtc (tbtc=4,5,9,14-tetraaza-benzo[b]triphenylene-11-carboxylic acid methyl ester) and its complexes [Ru(phen)2(tbtc)]2+ (1) (phen=1,10-phenanthroline) and [Ru(2,9-dmp)2(tbtc)]2+ (2) (2,9-dmp=2,9-dimethyl-1,10-phenanthroline) were synthesized and characterized by element analysis, MS, and 1H NMR. The DNA binding properties of both complexes to calf thymus DNA (CT-DNA) were investigated by different spectrophotometric methods and viscosity measurements. The results suggest that both complexes bind to DNA via an intercalative mode, and the DNA binding affinity of complex 1 is much greater than that of complex 2. This difference in binding affinity probably was caused by the different ancillary ligands. Also, when irradiated at 365 nm, complex 1 was found to be a more-effective DNA-cleaving agent than complex 2.     

新型钌(Ⅱ)多吡啶配合物与DNA作用的黏度法研究

用黏度法系统地研究了新型钌(Ⅱ)多吡啶配合物与DNA的相互作用.结果显示:在一定程度上增大配体的刚性平面,将增大钌(Ⅱ)多吡啶配合物插入DNA碱基对的能力,但当配体的刚性平面太大时,却又由于位阻作用而阻碍了配体对DNA的插入;能够使配体平面性增强的分子内氢键的形成,有利于配合物对DNA的插入作用;配体中引入较大体积的取代基而引起的配体芳环间的扭转,将导致钌(Ⅱ)多吡啶配合物只能“部分地”或“非经典地”插入DNA.     

毛细管电泳法高效筛选8-氧代鸟嘌呤DNA糖基化酶的核酸适配体

8-氧代鸟嘌呤DNA糖基化酶(OGG1)是人体中重要的功能蛋白,在修复DNA氧化性损伤过程中起关键作用。氧化应激等引起的氧化损伤易导致炎症反应的发生,对OGG1的抑制可以一定程度上起到缓解作用;对癌细胞OGG1的抑制有望作为癌症治疗的新方法。目前的研究多集中于小分子对OGG1功能的影响和调控,而OGG1的适配体筛选尚未见报道。作为功能配体,适配体具有合成简单、高亲和力及高特异性等优点。该文筛选了OGG1的核酸适配体,结合毛细管电泳高效快速的优点建立了两种基于毛细管电泳-指数富集进化(CE-SELEX)技术的筛选方法:同步竞争法和多轮筛选法。同步竞争法利用单链结合蛋白(SSB)与核酸库中单链核酸的强结合能力,与目标蛋白OGG1组成竞争体系,并通过增加SSB浓度来增加竞争筛选压力,以去除与OGG1弱结合的核酸序列,一步筛选即可获得与OGG1强结合的核酸序列。多轮筛选法在相同孵育条件和电泳条件下,经3轮筛选获得OGG1的核酸适配体。比较两种筛选方法的筛选结果,筛选结果中频次最高的3条候选核酸适配体序列一致,其解离常数(K_D)值在1.71~2.64 μmol/L之间。分子对接分析结果表明候选适配体1(Apt 1)可能与OGG1中具有修复氧化性损伤功能的活性口袋结合。通过对两种筛选方法的对比,证明同步竞争法更加快速高效,对其他蛋白核酸适配体筛选方法的选择具有一定的指导意义。得到的适配体有望用于OGG1功能调控,以抑制其修复功能。     

Comparison of mass spectrometry and other techniques for probing interactions between metal complexes and DNA

Electrospray ionization mass spectrometry (ESI-MS) was used to study the binding interactions of two series of ruthenium complexes, [Ru(phen) 2L] (2+) and [RuL' 2(dpqC)] (2+), to a double stranded DNA hexadecamer, and derive orders of relative binding affinity. These were shown to be in good agreement with orders of relative binding affinity derived from absorption and circular dichroism (CD) spectroscopic examination of the same systems and from DNA melting curves. However, the extent of luminescence enhancement caused by the addition of DNA to solutions of the ruthenium complexes showed little correlation with orders of binding affinity derived from ESI-MS or any of the other techniques. Overall the results provide support for the validity of using ESI-MS to investigate non-covalent interactions between metal complexes and DNA.     

DNA cleavage induced by photoexcited antimalarial drugs: a photophysical and photobiological study

The interactions and the photosensitizing activity of three antimalarial drugs quinine (Q), mefloquine (MQ) and quinacrine (QC) toward DNA was studied. Evidences obtained by absorption and emission spectroscopy and by linear dichroism measurements indicate that these derivatives bind the macromolecule with a high affinity (binding constants Ka approximately 10(5) M(-1)). The absorption characteristics of the drugs changed markedly by addition of DNA and their fluorescence was quenched with rate constants higher than that of diffusion. The geometry of binding involves predominantly the intercalation into the double helix. The DNA photocleavage properties of antimalarials was investigated using plasmid DNA as a model, at different [drug]/ [DNA] ratios. The results indicate that mainly MQ and Q are able to induce significant photodamage to DNA. In particular the marked effect of the former drug is evidenced after treatment of photosensitized DNA by two base excision repair enzymes, formamydo-pyrimidine glycosilase (Fpg) and Endonuclease III (Endo III). From a mechanistic point of view, experiments carried out in different experimental conditions indicate that these drugs photoinduce DNA damage through singlet oxygen and/or radical cation production. These findings are further supported by the determination of two photoproducts of 2'-deoxyguanosine, which are diagnostic for Type I and Type II pathways, namely 2,2-diamino(2-deoxy-beta-D-erythro-pentofuranosyl)-4-amino-5(2H)-oxazolone and (R,S)4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine (4-OH-8-oxo-dGuo). Laser flash photolysis experiments carried out in the presence of DNA indicates that the excitation produces mainly the triplet state for Q and the triplet and radical cation for QC. Moreover the singlet and triplet states and radical cations of the drugs are quenched by 2'-deoxyguanosine monophosphate. The absorbances of these transients decrease with increasing DNA concentration.     

Capillary electrophoresis as a probe of enantiospecific interactions between photoactive transition metal complexes and DNA

Recently, we have demonstrated the capacity to separate chiral transition metal (TM) complexes of the type [M(diimine)(3)](n+) using CE buffers containing chiral tartrate salts. In separate work, several chromium(III)-tris-diimine complexes in particular have been shown to bind enantioselectively with calf-thymus (CT) DNA, and a qualitative assessment of the relative strength and enantiospecificity of this interaction is of significant interest in the characterization of these complexes as potential DNA photocleavage agents. Here, we describe two convenient approaches to investigate such binding behavior using chiral CE. For complexes with lower DNA affinities exhibiting primarily surface binding, DNA itself is used as the chiral resolving agent in the electrophoretic buffer. In this approach, resolution of the TM complexes into their Lambda and Delta isomers is achieved with the isomer eluting later exhibiting superior binding affinity toward DNA. For more strongly bound TM complexes containing ligands known to intercalate with DNA, the [Cr(diimine)(3)](3+) complexes are preincubated with oligonucleotide and subsequently enantiomerically resolved in a dibenzoyl-L-tartrate buffer system that facilitates analysis of the unbound TM species only. Differences in isomer binding affinity are distinguished by the relative peak areas of the Lambda- and Delta-isomers, and relative binding strengths of different complexes can be inferred from comparison of the total amount of unbound complex at equivalent DNA/TM ratios.     

含平面胺配体的反式二价钯配合物与DNA碱基的作用

含大的平面胺配体的二价钯金属配合物在当前的抗肿瘤药物设计中代表着一类具有重要发展前途的先导结构. 由于大的平面胺配体具有较大的空间位阻, 目前主要的问题是这类化合物能否和DNA碱基结合形成单功能和双功能加合物. 我们采用密度泛函理论和等电聚焦连续极化(IEF-PCM)溶剂化模型研究了trans-PdCl2L2(L:2-羟基吡啶)的钯配合物与DNA碱基的作用. 该化合物与DNA形成单功能和双功能加合物反应的活化自由能均低于铂类抗肿瘤药. 所有反应在水溶液中均为放热反应. 结果表明, 这一大的平面胺配体不会阻碍该化合物与DNA碱基形成双功能加合物, 而且该化合物与DNA的单功能和双功能结合的速率会大于铂类化合物.     

新型多吡啶配体的设计及其对配合物与DNA作用机制的影响

近年来 ,以钌 ( )多吡啶配合物为探针研究 DNA的结构已成为生物无机化学领域中的一个热点[1,2 ] .这些配合物由于热力学稳定性好 ,光化学和光物理信息丰富 ,在研究 DNA内部的电子转移和Fig.1　 Structures of the ligandsDNA的结构识别等方面均有重要的作用[3～ 7] .在配合物与 DNA的相互作用中 ,配合物的形状、大小以及中心离子电荷等都有一定的影响[8] ,其中 ,配合物的形状起着至关重要的作用 ,与 DNA的形状匹配的配合物与DNA的结合较强 .这些配合物中通常含有平面性较大的芳香环配体 ,可插入到 DNA的碱基对之间 ,并与 DNA具有…