Formation of cis-diamminedichloroplatinum(II) 1,2-intrastrand cross-links on DNA is flanking-sequence independent

Mapping of cis-diamminedichloroplatinum(II) (cis-DDP, cisplatin) DNA adducts over >3000 nucleotides was carried out using a replication blockage assay. The sites of inhibition of modified T4 DNA polymerase, also referred to as stop sites, were analyzed to determine the effects of local sequence context on the distribution of intrastrand cisplatin cross-links. In a 3120 base fragment from replicative form M13mp18 DNA containing 24.6% guanine, 25.5% thymine, 26.9% adenine and 23.0% cytosine, 166 individual stop sites were observed at a bound platinum/nucleotide ratio of 1-2 per thousand. The majority of stop sites (90%) occurred at G(n>2) sequences and the remainder were located at sites containing an AG dinucleotide. For all of the GG sites present in the mapped sequences, including those with Gn(>)2, 89% blocked replication, whereas for the AG sites only 17% blocked replication. These blockage sites were independent of flanking nucleotides in a sequence of N(1)G*G*N(2) where N(1), N(2) = A, C, G, T and G*G* indicates a 1,2-intrastrand platinum cross-link. The absence of long-range sequence dependence was confirmed by monitoring the reaction of cisplatin with a plasmid containing an 800 bp insert of the human telomere repeat sequence (TTAGGG)(n). Platination reactions monitored at several formal platinum/nucleotide ratios or as a function of time reveal that the telomere insert was not preferentially damaged by cisplatin. Both replication blockage and telomere-insert plasmid platination experiments indicate that cisplatin 1,2-intrastrand adducts do not form preferentially at G-rich sequences in vitro.     

Flanking bases influence the nature of DNA distortion by platinum 1,2-intrastrand (GG) cross-links

The differences in efficacy and molecular mechanisms of platinum anti-cancer drugs cisplatin (CP) and oxaliplatin (OX) are thought to be partially due to the differences in the DNA conformations of the CP and OX adducts that form on adjacent guanines on DNA, which in turn influence the binding of damage-recognition proteins that control downstream effects of the adducts. Here we report a comprehensive comparison of the structural distortion of DNA caused by CP and OX adducts in the TGGT sequence context using nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations. When compared to our previous studies in other sequence contexts, these structural studies help us understand the effect of the sequence context on the conformation of Pt-GG DNA adducts. We find that both the sequence context and the type of Pt-GG DNA adduct (CP vs. OX) play an important role in the conformation and the conformational dynamics of Pt-DNA adducts, possibly explaining their influence on the ability of many damage-recognition proteins to bind to Pt-DNA adducts.     

Effects of 1,3-chelation induced by cis-diamminedichloroplatinum(II) on the stability of DNA duplexes.

Several 1,3-intra-strand cross-linked decadeoxynucleotide duplexes, modified with cis-diamminedichloroplatinum(II) (cis-DDP), and their base substitution analogues at the complementary site to the intervening base of the coordination sites, were synthesized and measured for UV-melting profiles to determine melting temperature (Tm) values. The results indicated the thermal stability of the oligonucleotide duplexes containing Pt-induced 1,3-intra-strand cross-linking did not depend on the kind of intervening base of the coordination site but rather on its complementary base. These results may explain the mutagenicity of cis-DDP from a chemical aspect.     

Cis-diamminedichloroplatinum(II) Induced Distortion of a Single and Double Stranded Deoxydecanucleosidenona-phosphate Studied by Nuclear Magnetic Resonance

Abstract

The structural distortion of a single- and a double-stranded decadeoxynucleotide upon binding of cis-PtCI₂(NH₃)₂ was studied by ¹H-NMR. After selective platination of d(T-C-T- C-G-G-T-C-T-C) (I) at the central d(-GpG-) site (resulting in I-Pt), several non-exchangeable base protons as well as H1′, H2′ H2″ and H3′ protons could be assigned by means of conventional NMR double-resonance techniques. Addition of the complementary decamer strand to I and I-Pt yielded the double-stranded III and III-Pt, respectively. All non-exchangeable base, H1′, and most of the H2′ and H2″ protons in the two double stranded compounds could be assigned using 2D-chemical shift correlation (COSY) and nuclear Overhauser enhancement (NOESY) techniques. The double stranded compound III appears to adopt a B-DNA like structure. Comparison of NOEs and proton-proton coupling constants in the d(-GpG-)·cisPt part in I-Pt and III-Pt reveals that their structure displays large similarity. Significant chemical shift changes (i.e, larger than 0.1 ppm) between III and III-Pt are restricted to the central four base pairs. It follows that the outer three base pairs, located on either side of the central four base pairs in III-Pt are likely to adopt a regular B-DNA type helix. The observed large upfield and downfield chemical shifts in the d(-CpGpG-) part of III with respect to III-Pt can be rationalized by describing the distortion of the double helix as a kink. A discussion of the observed physical effects upon platination of a double-stranded oligonucleotide is presented.     

Melting of Cross-Linked DNA V. Cross-Linking Effect Caused by Local Stabilization of the Double Helix

Abstract

DNA interstrand cross-links are usually formed due to bidentate covalent or coordination binding of a cross-linking agent to nucleotides of different strands. However interstrand linkages can be also caused by any type of chemical modification that gives rise to a strong local stabilization of the double helix. These stabilized sites conserve their helical structure and prevent local and total strand separation at temperatures above the melting of ordinary AT and GC base pairs. This local stabilization makes DNA melting fully reversible and independent of strand concentration like ordinary covalent interstrand cross-links. The stabilization can be caused by all the types of chemical modifications (interstrand cross-links, intrastrand cross-links or monofunctional adducts) if they give rise to a strong enough local stabilization of the double helix. Our calculation demonstrates that an increase in stability by 25 to 30 kcal in the free energy of a single base pair of the double helix is sufficient for this “cross-linking effect” (i.e. conserving the helicity of this base pair and preventing strand separation after melting of ordinary base pairs). For the situation where there is more then one stabilized site in a DNA duplex (e.g., 1 stabilized site per 1000 bp), a lower stabilization per site is sufficient for the “cross-linking effect” (18–20 kcal). A substantial increase in DNA stability was found in various experimental studies for some metal-based anti-tumor compounds. These compounds may give rise to the effect described above. If ligand induced stabilization is distributed among several neighboring base pairs, a much lower minimum increase per stabilized base pair is sufficient to produce the cross-linking effect (1 bp- 24.4 kcal; 5 bp- 5.3 kcal; 10 bp- 2.9 kcal, 25 bp- 1.4 kcal; 50 bp- 1.0 kcal). The relatively weak non-covalent binding of histones or protamines that cover long regions of DNA (20–40 bp) can also cause this effect if the salt concentration of the solution is sufficiently low to cause strong local stabilization of the double helix. Stretches of GC pairs more than 25 bp in length inserted into poly(AT) DNA also exhibit properties of stabilizing interstrand cross-links.     

Temperature Dependence of the DNA Double Helix at the Nanoscale: Structure,Elasticity, and Fluctuations

Biological organisms exist over a broad temperature range of −15°C to +120°C, where many molecular processes involving DNA depend on the nanoscale properties of the double helix. Here, we present results of extensive molecular dynamics simulations of DNA oligomers at different temperatures. We show that internal basepair conformations are strongly temperature-dependent, particularly in the stretch and opening degrees of freedom whose harmonic fluctuations can be considered the initial steps of the DNA melting pathway. The basepair step elasticity contains a weaker, but detectable, entropic contribution in the roll, tilt, and rise degrees of freedom. To extend the validity of our results to the temperature interval beyond the standard melting transition relevant to extremophiles, we estimate the effects of superhelical stress on the stability of the basepair steps, as computed from the Benham model. We predict that although the average twist decreases with temperature in vitro, the stabilizing external torque in vivo results in an increase of ∼1°/bp (or a superhelical density of Δσ ?  + 0.03) in the interval 0–100°C. In the final step, we show that the experimentally observed apparent bending persistence length of torsionally unconstrained DNA can be calculated from a hybrid model that accounts for the softening of the double helix and the presence of transient denaturation bubbles. Although the latter dominate the behavior close to the melting transition, the inclusion of helix softening is important around standard physiological temperatures.     

Temperature Dependence of the DNA Double Helix at the Nanoscale: Structure,Elasticity, and Fluctuations

Biological organisms exist over a broad temperature range of −15°C to +120°C, where many molecular processes involving DNA depend on the nanoscale properties of the double helix. Here, we present results of extensive molecular dynamics simulations of DNA oligomers at different temperatures. We show that internal basepair conformations are strongly temperature-dependent, particularly in the stretch and opening degrees of freedom whose harmonic fluctuations can be considered the initial steps of the DNA melting pathway. The basepair step elasticity contains a weaker, but detectable, entropic contribution in the roll, tilt, and rise degrees of freedom. To extend the validity of our results to the temperature interval beyond the standard melting transition relevant to extremophiles, we estimate the effects of superhelical stress on the stability of the basepair steps, as computed from the Benham model. We predict that although the average twist decreases with temperature in vitro, the stabilizing external torque in vivo results in an increase of ∼1°/bp (or a superhelical density of Δσ?+0.03$\text{Δ}\sigma ?+0.03$) in the interval 0–100°C. In the final step, we show that the experimentally observed apparent bending persistence length of torsionally unconstrained DNA can be calculated from a hybrid model that accounts for the softening of the double helix and the presence of transient denaturation bubbles. Although the latter dominate the behavior close to the melting transition, the inclusion of helix softening is important around standard physiological temperatures.     

Distortions induced in double-stranded oligonucleotides by the binding of cis- or trans-diammine-dichloroplatinum(II) to the d(GTG) sequence.

Conformational changes induced in double-stranded oligonucleotides by the binding of trans- or cis-diamminedichloro platinum(II) to the d(GTG) sequence have been characterized by means of melting temperatures, electrophoretic migrations in non-denaturing polyacrylamide gels, reactivities with the artificial nuclease Phenanthroline-copper and with chemical probes. The cis-platinum adduct behaves more as a centre of directed bend than as a hinge joint, the induced bend angle being of the order of 25-30 degrees. The double helix is locally denatured over 2 base pairs (corresponding to the platinated 5'G residue and the central T residue) and is distorted over 4-5 base pairs. The trans-platinum adduct behaves also more as a centre of directed bend than as a hinge joint, the induced bend angle being of the order of 60 degrees. The double helix is locally denatured over 4 base pairs (corresponding to the immediately 5'T residue adjacent to the adduct and to the three base residues of the adduct). Both the cis- and trans-platinum adducts decrease the thermal stability of the double helix.     

Conformational Isomerizations of the Poly(dA-dT) and Poly(amino2dA-dT) Duplexes Involving the Unusual X-DNA Double Helix: A Fourth Derivative Spectrophotometric Study

Abstract

Fourth derivative spectrophotometry has been applied to monitor conformational isomerizations of polynucleotides for the first time. The transitions studied have been the B-A and A-X isomerizations of poly(dA-dT) and the B-X one of poly(amino²dA-dT). Parameters obtained from the fourth derivative spectra have been used to follow these conformational changes. The A form of poly(dA-dT) has been characterized by a new fourth derivative peak at 293.0 nm which can be associated to interstrand adenine-adenine interactions. Furthermore, some of the fourth derivative peaks in the long wavelength region (270–310 nm) can be related to stacking interactions present in the polynucleotide double helices. The tentative assignment of these peaks, particularly that at 299.0 nm in the derivative spectra of poly(amino²dA-dT), to n→π^* electronic transitions is discussed.     

Left-Handed Intercalated DNA Double Helix: Rendezvous of Ethidium and Actinomycin D in the Z-Helical Conformation Space

Abstract

It is now very well recognized that the DNA double helix is conformationally pluralistic and that this flexibility is derived from internal motions due to backbone torsions. But what is less apparent is that such internal motions can occur in a correlated fashion and express themselves in a wide variety of structural motifs and phenomena. For example, flexibility inherent in the DNA molecule can lead to a family of Z-DNA, LZ1 and LZ2 being the two extremes and correlated internal motion can cause LZ1?LZ2 transition. More interestingly, such motions manifest themselves as breathing modes on the DNA lattice resulting in the sequence specific intercalation sites. Following a detailed stereochemical analyses we observed that the intercalation site for ethidium is located at the dCpdG sequence of the intercalated LZ1 helix (LZ1*) while that for actinomycin D is located at the dGpdC sequence of the intercalated LZ2 helix (LZ2*). From the stereochemistry of the drug binding we make experimentally testable predictions which are in fact supported by a few recent experimental studies. These studies also show that a left-handed intercalated B-DNA model is a viable intermediate in the Z to B transition which can hold the drug with binding energy comparable to that of the intercalated right-handed B-DNA.     

Comparison of the Structures of the Metal-thiolate Binding Site in Zn(II)-, Cd(II)-, and Hg(II)-Metallothioneins Using Molecular Modeling Techniques

Abstract

The first fully energy-minimized structures for a series of structurally related metal complexes of the important mammalian metal binding protein metallothionein are described. The structures were calculated based on structural information obtained from existing spectroscopic and crystallographic data, and minimized using molecular mechanics (MM2) techniques. A two domain structure, with stoichiometrics of M(II)₃?(S_cys)₉ and M(II)₄?(S_cys)₁₁ where M = zinc(II), cadmium(II), and mercury(II), was assembled and minimized. The resultant three-dimensional structure closely resembled that of rat liver Cd₅Zn₂?MT 1 obtained by analysis of x-ray diffraction data [A. H. Robbins, D. E. McRee, M. Williamson, S. A. Collett, N. H. Xuong, W. F. Furey, B. C. Wang and C. D. Stout, J. Mol. Biol. 221, 1269–1293 (1991)]. Minimized structures for Zn₇?MT, Cd₇?MT, and Hg₇?MT are reported. Deep crevices that expose the metal-thiolate clusters are seen in each structure. However, for the mercury-containing protein, much of the mercury-thiolate structure is visible and it is proposed that this provides access for extensive interaction between solvent water molecules and the mercury(II), resulting in the observed distortion away from tetrahedral geometry for Hg₇MT. Volume calculations are reported for the protein metallated with 7 Zn(II), Cd(II), or Hg(II). A series of structural changes calculated for the step-wise isomorphous replacement of Zn(II) by Cd(II) and Hg(II) in the Zn₄S₁₁ α domain are shown.     

Probes for Double Helical DNA Sequence Information: Molecular Mechanics Study of a Proposed Model

Abstract

The biological activity of many molecules which bind to double helical DNA is related to the sequence specificity of their binding. The development of new substances of this type is challenging; a general solution to the problem does not exist. A new mechanism for small molecule-duplex DNA interaction termed intercalative displacement is proposed. The approach is promising for the design of new substances which will recognize sequence information on DNA Molecular mechanics calculations in the absence of solvent and counterions predict that the proposed model is structurally and energetically closely related to the well known process of standard intercalation. The implications of these calculations for experimental studies are discussed.     

The Role of Glutathione in Protection against DNA Damage Induced by Rifamycin SV And Copper(II) Ions

Incubation of calf thymus DNA in the presence of rifamycin SV induces a decrease in the absorbance of DNA at 260 nm. The effect, was found to be proportional to the antibiotic concentration and enhanced by copper(II) ions. In the presence of rifamycin SV and copper(II), a significant increase in thiobarbituric acid-reactive (TBA-reactive) material is also observed. This effect is inhibited to different degrees by the following antioxidants: catalase 77%; thiourea 72%; glutathione (GSH) 62%; ethanol 52%; and DMSO 34%, suggesting that both hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH·) are involved in DNA damage. Rifamycin SV-copper(II) mixtures were also found to induce the production of peroxidation material from deoxyribose and, in this case, glutathione and ethanol were the most effective antioxidant substrates with inhibition rates of 91% and 88% respectively.

Electrophoretic studies show that calf thymus DNA becomes damaged after 20 min. incubation in the presence of both agents together and that the damaged fragments run with migration rates similar to those obtained by the metal chelating agent 1,10-phenanthroline. Normal DNA electrophoretic pattern was found to be preserved by catalase, and GSH at physiological concentrations and by thiourea. No protection is observed in the presence of ethanol or DMSO. The results obtained indicate the involvement of different reactive species in the degradation process of DNA due to rifamycin SV-copper(II) complex and emphasize the role of reduced glutathione as an oxygen free radical scavenger.     

A Small Molecule Inhibitor of Monoubiquitinated Proliferating Cell Nuclear Antigen (PCNA) Inhibits Repair of Interstrand DNA Cross-link,Enhances DNA Double Strand Break,and Sensitizes Cancer Cells to Cisplatin

Small molecule inhibitors of proliferating cell nuclear antigen (PCNA)/PCNA interacting protein box (PIP-Box) interactions, including T2 amino alcohol (T2AA), inhibit translesion DNA synthesis. The crystal structure of PCNA in complex with T2AA revealed that T2AA bound to the surface adjacent to the subunit interface of the homotrimer of PCNA in addition to the PIP-box binding cavity. Because this site is close to Lys-164, which is monoubiquitinated by RAD18, we postulated that T2AA would affect monoubiquitinated PCNA interactions. Binding of monoubiquitinated PCNA and a purified pol η fragment containing the UBZ and PIP-box was inhibited by T2AA in vitro. T2AA decreased PCNA/pol η and PCNA/REV1 chromatin colocalization but did not inhibit PCNA monoubiquitination, suggesting that T2AA hinders interactions of pol η and REV1 with monoubiquitinated PCNA. Interstrand DNA cross-links (ICLs) are repaired by mechanisms using translesion DNA synthesis that is regulated by monoubiquitinated PCNA. T2AA significantly delayed reactivation of a reporter plasmid containing an ICL. Neutral comet analysis of cells receiving T2AA in addition to cisplatin revealed that T2AA significantly enhanced formation of DNA double strand breaks (DSBs) by cisplatin. T2AA promoted colocalized foci formation of phospho-ATM and 53BP1 and up-regulated phospho-BRCA1 in cisplatin-treated cells, suggesting that T2AA increases DSBs. When cells were treated by cisplatin and T2AA, their clonogenic survival was significantly less than that of those treated by cisplatin only. These findings show that the inhibitors of monoubiquitinated PCNA chemosensitize cells by inhibiting repair of ICLs and DSBs.     

Study of the interaction of DNA with cisplatin and other Pd(II) and Pt(II) complexes by atomic force microscopy.

Modifications in the structure of a 260 bp DNA (hlyM) fragment from Escherichia coli caused by interaction with Pd(II) and Pt(II) complexes were studied. Cisplatin and transplatin [cis- and trans-PtCl2(NH3)2 respectively], Pt2Cl2(Spym)4 (Spym = 2-mercaptopyrimidine anion), Pd-famotidine and Pt-famotidine were incubated with DNA for 24 h at 37 degrees C and then observed with an atomic force microscope. Atomic force microscopy (AFM) provides the opportunity for nanometer resolution in research on the interaction between nucleic acids and metal complexes. The complexes induced noticeable changes in DNA topography according to their different characteristics and structure. In the case of cisplatin a shortening in DNA strands was observed. Transplatin and Pt2Cl2(Spym)4 caused shortening and compaction, whilst an aggregation of two strands was observed for the Pt-famotidine compound but not for the Pd-famotidine compound or the metal-free famotidine.     

The Flexibility of the Nucleic Acids: (II) The Calculation of Internal Energy and Applications to Mononucleotide Repeat DNA

Abstract

Results concerning the flexibility of mononucleotide repeat DNA are presented using a novel methodology, denoted “SIR”, to describe continuous changes in the structure of the nucleic acid. This methodology, combined with internal energy calculations and analytical energy gradients allows us to determine optimal conformations of poly(dG)?poly(dC) and poly (dA)?poly(dT) in both the A and B forms, taking into account the influence of the solvent medium and explicit counterions. Subsequently, several different types of distorsion of these optimal structures are investigated. It is shown that excellent correlation with experimental results concerning coupled changes in structural variables is obtained and several new correlations are also detected.     

Mechanisms Modeling the Double Rotation of the Elytra in Beetles (Coleoptera)

We recorded transient movements,i.e.opening and closing,of beetle elytra.The beetles were tethered from below and filmed under a skew mirror; two markers were glued on each elytron at the apex and at the base.Body-fixed 3D traces of the apical and basal markers were reconstructed.The trace of the basal marker was,as a rule,non-parallel to the apical trace.The costal edge of the elytron uniformly supinated in the course of adduction of the apical marker.We found two essential attributes of double rotation:(1) the elytron to body articulation is approximately a spherical mechanism; (2) transient opening and closing possess single degree of freedom.The double rotation was modeled with two mechanisms:(1) a flexagon model of the Haas and Wootton's type simulated the elytral movement relative to the movement of one facet of the flexagon; (2) a screw and nut model provided traces as two sectors of a helical thread,one sector was phase shifted with respect to other one.Screw guideways in a spherical mechanism give rise to discrepancies.Exact solution for a spherical mechanism with two guideways was proposed.The modeling revealed the attribute (3):the elytron is actuated by two linked but differently directed drives.Experimental investigations on the elytron to body articulation may be oriented at search of those mechanisms.