DNA compaction by mononuclear platinum cancer drug cisplatin and the trisplatinum anticancer agent BBR3464: Differences and similarities

Cisplatin, a mononuclear platinum compound, which is known as a cancer drug for long time, can exhibit considerable side effects and is also not effective in many types of cancer. Therefore, the alternative platinum anticancer agents that can act at a much lower dose limit compared to the dose relevant for cisplatin treatment have been searched for. BBR3464, a trinuclear platinum compound, is found to exhibit cytotoxic effects at 10 to 1000 times lower dose limit, even in cisplatin-resistant cancer cells. The primary cellular target for cisplatin and BBR3464 is thought to be DNA. Herein, we report the nature of DNA structural changes that are induced by cisplatin and BBR3464, considering the same DNA sequence and similar sample deposition methods for comparison purpose. We have applied high-resolution atomic force microscopy (AFM) in order to obtain an idea about the molecular basis of BBR3464's effectiveness at the lower dose limit. We show from the molecularly resolved AFM images that both the compounds can compact the whole dsDNA molecules, though the degree of compaction in case of BBR3464 treatment is significantly higher. Furthermore, local compaction in terms of loop structure formation could be induced by both BBR3464 and cisplatin, though BBR3464 generated microloops and macroloops both, whereas cisplatin could generate primarily the microloops. It is a significant observation that BBR3464 could induce relatively drastic DNA structural changes in terms of loop formation as well as overall DNA compaction at a molar ratio, which is 50 times less than that applied for cisplatin treatment. Implications of such structural changes in cytotoxic effects of the platinum anticancer agents will be mentioned.     

A close encounter with DNA

Atomic force microscopy, associated with surface science, has the potential to resolve the secondary structure of DNA in liquid form with unusually high resolution and with unprecedented accuracy.     

The Bacillus subtilis DnaD protein: a putative link between DNA remodeling and initiation of DNA replication

The Bacillus subtilis DnaD protein is an essential protein and a component of the oriC and PriA primosomal cascades, which are responsible for loading the main replicative ring helicase DnaC onto DNA. We present evidence that DnaD also has a global DNA architectural activity, assembling into large nucleoprotein complexes on a plasmid and counteracting plasmid compaction in a manner analogous to that recently seen for the histone-like Escherichia coli HU proteins. This DNA-remodeling role may be an essential function for initiation of DNA replication in the Gram +ve B. subtilis, thus highlighting DnaD as the link between bacterial nucleoid reorganization and initiation of DNA replication.     

Structure, force, and energy of a double-stranded DNA oligonucleotide under tensile loads

The end-to-end stretching of a duplex DNA oligonucleotide has been studied using potential of mean force (PMF) calculations based on molecular dynamics (MD) simulations and atomic force microscopy (AFM) experiments. Near quantitative agreement between the calculations and experiments was obtained for both the extension length and forces associated with strand separation. The PMF calculations show that the oligonucleotide extends without a significant energetic barrier from a length shorter than A-DNA to a length 2.4 times the contour length of B-DNA at which the barrier to strand separation is encountered. Calculated forces associated with the barrier are 0.09±0.03 nN, based on assumptions concerning tip and thermal-activated barrier crossing contributions to the forces. Direct AFM measurements show the oligonucleotide strands separating at 2.6±0.8 contour lengths with a force of 0.13±0.05 nN. Analysis of the energies from the MD simulations during extension reveals compensation between increases in the DNA-self energy and decreases in the DNA-solvent interaction energy, allowing for the barrierless extension of DNA beyond the canonical B form. The barrier to strand separation occurs when unfavorable DNA interstrand repulsion cannot be compensated for by favorable DNA-solvent interactions. The present combination of single molecule theoretical and experimental approaches produces a comprehensive picture of the free energy surface of biological macromolecular structural transitions. Received: 2 June 1998 / Revised version: 25 January 1999 / Accepted: 11 February 1999     

Characterization of condensed plasmid DNA models for studying the direct effect of ionizing radiation

We have examined the changes in physical properties of aqueous solutions of the plasmid pUC18 that take place on the addition of the cationic oligopeptide penta-arginine. An increase in sedimentation rate and static light scattering, and changes in the nucleic acid CD spectrum all suggest that this ligand acts to condense the plasmid. Dynamic light scattering suggests the hydrodynamic radii of the condensate particles are a few micrometers, ca. 50-fold larger than that of the monomeric plasmid. Condensation of the plasmid also produces a ca. 100-fold decrease in the strand break yield produced by gamma irradiation. This extensive protection against reactive intermediates in the bulk of the solution implies that condensed plasmid DNA may offer a model system with which to study the direct effect of ionizing radiation (ionization of the DNA itself). The use of peptide ligands as condensing agents in this application is attractive because the derivatives of several amino acids (particularly tryptophan and tyrosine) have been shown to modify the radiation chemistry of DNA extensively.     

Visualization of alkali-denatured supercoiled plasmid DNA by atomic force microscopy

To study the alkali denaturation of supercoiled DNA, plasmid pBR322 was treated with gradient concentrations of NaOH solution. The results of gel electrophoresis showed that the alkali denaturation of the supercoiled DNA occurred in a narrow range of pH value (12.88-12.90). The alkali-denatured supercoiled DNA ran, as a sharp band, faster than the supercoiled DNA. The supercoiled plasmid DNA of pBR322, pACYC184 and pJGX15A were denatured by NaOH, and then visualized by atomic force microscopy. Compared with the supercoiled DNA, the atomic force microscopy images of the alkali-denatured supercoiled DNA showed rough surface with many kinks, bulges on double strands with inhomogeneous diameters. The apparent contour lengths of the denatured DNA were shortened by 16%, 16% and 50% for pBR322, pACYC184 and pJGX15A, respectively. All evidence suggested that the alkali-denatured supercoiled DNA had a stable conformation with unregistered, topologically constrained double strands and intrastrand secondary structure.     

Studying the effect of a charged surface on the interaction of bleomycin with DNA using an atomic force microscope

The cleavage of DNA caused by the antitumoral drug bleomycin has been investigated using atomic force microscopy (AFM). This work deals with the effect that adsorbing DNA onto a positively- or negatively-charged surface has on the double-strand cleavage of DNA by Fe(III)/bleomycin. Quantitative analysis of the number of breaks per DNA molecule, in bulk and at the surface of the mica substrate, has been performed by analyzing AFM images. It turns out that the cleavage of DNA is strongly inhibited by a positively-charged surface. Our experiments can be interpreted using a simple electrostatic model. This paper is a first step in the study of DNA accessibility to ligand such as bleomycin, using AFM in liquids.Olivier Piétrement and David Pastré have contributed equally to the work.     

Compaction of single supercoiled DNA molecules adsorbed onto amino mica

A model of possible conformational transitions of supercoiled DNA in vitro in the absence of proteins under the conditions of increasing degree of compaction was developed. A 3993-bp pGEMEX supercoiled DNA immobilized on various substrates (freshly cleaved mica, standard amino mica, and modified amino mica with a hydrophobicity higher than that of standard amino mica) was visualized by atomic force microscopy in air. On the modified amino mica, which has an increased density of surface positive charges, single molecules with an extremely high degree of compaction were visualized in addition to plectonemic DNA molecules. As the degree of DNA supercoiling increased, the length of the first-order superhelical axis of molecules decreased from 570 to 370 nm, followed by the formation of second-and third-order superhelical axes about 280 and 140 nm long, respectively. The compaction of molecules ends with the formation of minitoroids about 50 nm in diameter and molecules of spherical shape. It was shown that the compaction of single supercoiled DNA molecules immobilized on amino mica to the level of minitoroids and spheroids is due to the shielding of mutually repulsing negatively charged phosphate groups of DNA by positively charged amino groups of the amino mica, which has a high charge density of its surface.     

Detection of cruciform extrusion in DNA by temperature-gradient gel electrophoresis

Repetitive sequences in DNA molecules, some of which are palindromic, tend to form stable cruciforms. These are frequently located in promoter regions of a specific operon and origin of replication. Temperature gradient gel electrophoresis can be used to distinguish among various supercoiled DNA topoisomers and to ascertain whether or not the cruciform motif has been extruded. In the current study, this technique is implemented for the first time to address the role of temperature in cruciform extrusion from plasmids.     

Insight into the cooperative DNA binding of the O6-alkylguanine DNA alkyltransferase

The O⁶-alkylguanine DNA alkyltransferase (AGT) is a highly conserved protein responsible for direct repair of alkylated guanine and to a lesser degree thymine bases. While specific DNA lesion-bound complexes in crystal structures consist of monomeric AGT, several solution studies have suggested that cooperative DNA binding plays a role in the physiological activities of AGT. Cooperative AGT–DNA complexes have been described by theoretical models, which can be tested by atomic force microscopy (AFM). Direct access to structural features of AGT–DNA complexes at the single molecule level by AFM imaging revealed non-specifically bound, cooperative complexes with limited cluster length. Implications of cooperative binding in AGT–DNA interactions are discussed.     

Fine mapping of inherent flexibility variation along DNA molecules. Validation by atomic force microscopy (AFM) in buffer

Curvature and flexibility are structural properties of central importance to genome function. However, due to the difficulties in finding suitable experimental conditions, methods for studying one without the interference of the other have proven to be difficult. We propose a new approach that provides a measure of inherent flexibility of DNA by taking advantage of two powerful techniques, X-ray crystallography and nuclear magnetic resonance. Both techniques are able to detect local curvature on DNA fragments but, while the first analyzes DNA in the solid state, the second works on DNA in solution. Comparison of the two data sets allowed us to calculate the relative contribution to flexibility of the three rotations and three translations, which relate successive base pair planes for the ten different dinucleotide steps. These values were then used to compute the variation of flexibility along a given nucleotide sequence. This allowed us to validate the method experimentally through comparisons with maps of local fluctuations in DNA molecule trajectory constructed from atomic force microscopy imaging in solution. We conclude that the six dinucleotide-step parameters defined here provide a powerful tool for the exploration of DNA structure and, consequently will make an important contribution to our understanding of DNA-sequence-dependent biological processes.     

The region ion sensitive field effect transistor, a novel bioelectronic nanosensor

A novel type of bioelectronic region ion sensitive field effect transistor (RISFET) nanosensor was constructed and demonstrated on two different sensor chips that could measure glucose with good linearity in the range of 0–0.6 mM and 0–0.3 mM with a limit of detection of 0.1 and 0.04 mM, respectively. The sensor is based on the principle of focusing charged reaction products with an electrical field in a region between the sensing electrodes. For glucose measurements, negatively charged gluconate ions were gathered between the sensing electrodes. The signal current response was measured using a low-noise pico ammeter (pA). Two different sizes of the RISFET sensor chips were constructed using conventional electron beam lithography. The measurements are done in partial volumes mainly restricted by the working distance between the sensing electrodes (790 and 2500 nm, respectively) and the influence of electrical fields that are concentrating the ions. The sensitivity was 28 pA/mM (2500 nm) and 830 pA/mM (790 nm), respectively. That is an increase in field strength by five times between the sensing electrodes increased the sensitivity by 30 times. The volumes expressed in this way are in low or sub femtoliter range. Preliminary studies revealed that with suitable modification and control of parameters such as the electric control signals and the chip electrode dimensions this sensor could also be used as a nanobiosensor by applying single enzyme molecule trapping. Hypotheses are given for impedance factors of the RISFET conducting channel.     

Atomic force microscopy captures the initiation of methyl-directed DNA mismatch repair

In Escherichia coli, errors in newly-replicated DNA, such as the incorporation of a nucleotide with a mis-paired base or an accidental insertion or deletion of nucleotides, are corrected by a methyl-directed mismatch repair (MMR) pathway. While the enzymology of MMR has long been established, many fundamental aspects of its mechanisms remain elusive, such as the structures, compositions, and orientations of complexes of MutS, MutL, and MutH as they initiate repair. Using atomic force microscopy, we—for the first time—record the structures and locations of individual complexes of MutS, MutL and MutH bound to DNA molecules during the initial stages of mismatch repair. This technique reveals a number of striking and unexpected structures, such as the growth and disassembly of large multimeric complexes at mismatched sites, complexes of MutS and MutL anchoring latent MutH onto hemi-methylated d(GATC) sites or bound themselves at nicks in the DNA, and complexes directly bridging mismatched and hemi-methylated d(GATC) sites by looping the DNA. The observations from these single-molecule studies provide new opportunities to resolve some of the long-standing controversies in the field and underscore the dynamic heterogeneity and versatility of MutSLH complexes in the repair process.     

Structural changes of DNA induced by mono- and binuclear cancer drugs

The structural features of the drug-DNA adducts resulted from treatment of DNA with the platinum based mononuclear drug cisplatin and the binuclear drug [{trans-PtCl(NH3)2}2H2N(CH2)4NH2]Cl2 or bis(platin) have been investigated by atomic force microscopy (AFM). Reduction in the contour length of the DNA fragments has been observed after cisplatin treatment while, compaction and aggregation are found to be the primary structural modifications following treatment with the binuclear drug. The intermolecular interaction upon bis(platin) treatment leads to observation of highly condense aggregates without a distinct sight of single isolated DNA molecule. These differences in drug binding indicate that unlike the mononuclear drug cisplatin, bis(platin) causes extensive interhelical/intermolecular cross-linking through its multiple linking sites. To our knowledge, this is the first report of a comparative AFM study to monitor the effects of a mono- and a binuclear platinum anti-cancer drug on DNA structure. These observations should provide clues towards explaining the distinct biological activities of the two drugs.     

Proteomic characterization of the cytotoxic mechanism of gold (III) porphyrin 1a, a potential anticancer drug

There has been increasing interest in the potential applications of gold (III) complexes as anticancer drugs with higher cytotoxicity and fewer side effects than existing metal anticancer drugs. Our previous findings demonstrated that gold (III) porphyrin 1a preferentially induced apoptosis in a cancer cell line (SUNE1). In this study, we identified differentially expressed proteins related to the drug's cytotoxic action by comparing the protein alterations induced by gold (III) porphyrin 1a and cisplatin treatments. Several clusters of altered proteins were identified, including cellular structure and stress-related chaperone proteins, proteins involved in reactive oxygen species and enzyme proteins, translation factors, proteins that mediate cell proliferation or differentiation, and proteins participating in the internal degradation systems. Our results indicated that multiple factors leading to apoptosis were involved in drug cytotoxicity in SUNE1 cells. The balance between pro-apoptotic and anti-apoptotic signals determined the final fate of cancer cells.     

细菌RNA结合蛋白Hfq的DNA压缩和复制作用研究进展

RNA结合蛋白(RNA-Binding Protein)Hfq是一种重要的细菌转录后调节因子,之前对Hfq的研究大多集中在该蛋白对小分子非编码RNA(Small Non-Coding RNA,sRNA)和mRNA的作用上.Hfq最典型的功能是促进sRNA与其靶标mRNA碱基配对,在转录后介导对RNA的稳定性和翻译的调控...     

原子力显微镜观测紫外线诱导的K562肿瘤细胞DNA损伤

利用彗星电泳检测出UVB、UVC短时间照射会使肿瘤细胞的DNA发生断裂,而长时间照射之后彗星电泳无法检测到碎片,推测可能是由于DNA分子交联的原因[1],国内外尚无定论.为了更直观的研究这种现象,提取了UVB,UVA照射后K562细胞的DNA,并调节到合适的浓度在原子力显微镜下观测.实验结果表明UVB对K562肿瘤细胞DNA损伤的影响呈现时间/剂量效应,较短时间照射主要产生DNA的链断裂,较长时间辐射则主要产生DNA链的交联.UVC对K562肿瘤细胞DNA的损伤大于UVB.UVC短时照射即可引起DNA的断裂和交联,较长时间辐射主要产生交联和一些断裂;长时间照射不但产生大量交联,同时有大量断裂产生,并发生凝缩和缠绕等结构破坏.