Modulation of MutS ATP-dependent functional activities by DNA containing a cisplatin compound lesion (base damage and mismatch)

DNA damage-dependent signaling by the DNA mismatch repair (MMR) system is thought to mediate cytotoxicity of the anti-tumor drug cisplatin through molecular mechanisms that could differ from those required for normal mismatch repair. The present study investigated whether ATP-dependent biochemical properties of Escherichia coli MutS protein differ when the protein interacts with a DNA oligonucleotide containing a GT mismatch versus a unique site specifically placed cisplatin compound lesion, a cisplatin 1,2-d(GpG) intrastrand cross-link with a mispaired thymine opposite the 3' platinated guanine. MutS exhibited substantial affinity for this compound lesion in hydrolytic and in non-hydrolytic conditions of ATP, contrasting with the normal nucleotide inhibition effect of mispair binding. The cisplatin compound lesion was also shown to stimulate poorly MutS ATPase activity to approach the hydrolysis rate induced by nonspecific DNA. Moreover, MutS undergoes distinct conformation changes in the presence of the compound lesion and ATP under hydrolytic conditions as shown by limited proteolysis. In the absence of MutS, the cisplatin compound lesion was shown to induce a 39 degrees rigid bending of the DNA double helix contrasting with an unbent state for DNA containing a GT mispair. Furthermore, an unbent DNA substrate containing a monofunctional adduct mimicking a cisplatin residue failed to form a persistent nucleoprotein complex with MutS in the presence of adenine nucleotide. We propose that DNA bending could play a role in MutS biochemical modulations induced by a compound lesion and that cisplatin DNA damage signaling by the MMR system could be modulated in a direct mode.     

Binding of NAD+ to pertussis toxin.

The equilibrium dissociation constant of NAD+ and pertussis toxin was determined by equilibrium dialysis and by the quenching of the protein's intrinsic fluorescence on titration with NAD+. A binding constant, Kd, of 24 +/- 2 microM at 30 degrees C was obtained from equilibrium dialysis, consistent with the previously determined value for the Michaelis constant, Km, of 30 +/- 5 microM for NAD+ (when the toxin is catalysing the ADP-ribosylation of water and of dithiothreitol). The intrinsic fluorescence of pertussis toxin was quenched by up to 60% on titration with NAD+, and after correction for dilution and inner filter effects, a Kd value of 27 microM at 30 degrees C was obtained, agreeing well with that found by equilibrium dialysis. The binding constants were measured at a number of temperatures using both techniques, and from this the enthalpy of binding of NAD+ to toxin was determined to be 30 kJ.mol-1, a typical value for a protein-ligand interaction. There is one binding site for NAD+ per toxin molecule.     

The effect of C-terminal helix stabilization on specific DNA binding by monomeric GCN4 peptides.

DNA binding by a 29-residue, monomeric, GCN4 basic region peptide, GCN4br, as well as by peptide br-C, a monomeric basic-region analogue that is helix stabilized at its C-terminal end by a Lys25. Asp29 side-chain lactam-bridged alanine-rich sequence, was studied at 25 C in an aqueous buffer containing 100 mm NaCl. Mixing of both peptides with duplex DNA containing the cAMP-responsive element (CRE) was accompanied by significant helix stabilization in the peptides, whereas mixing of the peptides with duplex DNA containing a scrambled CRE site was not. Peptide NBD-br-C was synthesized as a fluorescent probe to evaluate these peptide-DNA interactions further. Quantitative analysis of the fluorescence quenching of peptide NBD-br-C by CRE half-site DNA indicated the formation of a 1:1 complex with a dissociation constant of 1.41 +/- 0.22 microm. Competitive displacement fluorescence assays of CRE half-site binding gave dissociation constants of 0.65 +/- 0.09 microm for peptide br-C and 3.9 +/- 0.5 microM for GCN4br, which corresponds to a free energy difference of 1.1 kcal/mol that is attributed to the helix stabilization achieved in peptide br-C. This result indicates that helix initiation by the alpha-helical leucine zipper dimerization motif in native bzip proteins, such as GCN4, contributes significantly to the affinity of basic region peptides for their recognition sites on DNA. Our fluorescence assay should also prove useful for determining dissociation constants for CRE binding by other GCN4 basic region analogues under equilibrium conditions and physiological salt concentrations.     

Biochemistry of terminal deoxynucleotidyltransferase (TdT): characterization and mechanism of inhibition of TdT by P1, P5-bis(5'-adenosyl) pentaphosphate

The catalysis of DNA synthesis by calf thymus terminal deoxynucleotidyltransferase (TdT) is strongly inhibited in the presence of Ap5A, while replicative DNA polymerases from mammalian, bacterial, and oncornaviral sources are totally insensitive to Ap5A addition. The Ap5A-mediated inhibition of TdT seems to occur via its interaction at both the substrate binding and primer binding domains as judged by classical competitive inhibition plots with respect to both substrate deoxynucleoside triphosphate (dNTP) and DNA primer and inhibition of ultraviolet light mediated cross-linking of substrate dNTP and oligomeric DNA primer to their respective binding sites. Further kinetic analyses of Ap5A inhibition revealed that the dissociation constant of the Ap5A-enzyme complex, with either substrate binding or primer binding domain participating in the complex formation, is approximately 6 times higher (Ki = 1.5 microM) compared to the dissociation constant (Ki = 0.25 microM) of the Ap5A-TdT complex when both domains are available for binding. In order to study the binding stoichiometry of Ap5A to TdT, an oxidized derivative of Ap5A, which exhibited identical inhibitory properties as its parent compound, was employed. The oxidation product of Ap5A, presumably a tetraaldehyde derivative, binds irreversibly to TdT when the inhibitor-enzyme complex is subjected to borohydride reduction. The presence of aldehyde groups in the oxidized Ap5A appeared essential for inhibitory activity since its reduction to alcohol via borohydride reduction or its linkage to free amino acids prior to use as an inhibitor rendered it completely ineffective.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of the size of the ligand molecule on the stability of the DNA double helix

The influence of ligand length on helix-coil transition parameters in the presence of different ligand concentrations has been considered theoretically. A decrease of this influence with an increase of ligand length when the binding constant has a constant value was shown. When binding free energy is proportional to the ligand length, i. e. when K = K0m (K-binding constant, m-ligand length) the length effect is unambigous. In the presence of low-ligand concentration the stability of DNA double helix increases with their length, whereas in the presence of high concentrations it decreases.     

Tetracyanonickelate probes the active site of sulfur-free rhodanese

Tetracyanonickelate (Ni(CN)4(2-)) was used as a probe for the active site of sulfur-free rhodanese (E) in physical and kinetic studies. Ni(CN)4(2-) quenches the intrinsic fluorescence as well as the fluorescence of enzyme-bound 2-anilinonaphthalene-8-sulfonic acid (2,8-ANS), an inhibitor that is competitive with respect to thiosulfate. A facile binding method based on centrifugation was developed to study Ni(CN)4(2-) binding to E. Binding studies performed using either of the electrophoretic variants A and B, fractionated by DE52 column chromatography, showed one high affinity Ni(CN)4(2-)-binding site in each species and additional weak sites on the more electropositive form A. The high affinity Ni(CN)4(2-) binding was corroborated by ultrafiltration binding (Kd = 3.95 +/- 0.35 microM), titration of intrinsic fluorescence (Kd = 1.8 +/- 0.11 microM), and displacement of enzyme-bound 2,8-ANS (Kd = 1.9 +/- 1.1 microM). A nonlinear least squares analysis of kinetic data collected under conditions used for the binding studies gave a Ni(CN)4(2-) inhibition constant of 21 microM. It is concluded that Ni(CN)4(2-) binds to sulfur-free rhodanese in solution near the active site as has been shown in x-ray crystal studies (Lijk, L. J., Kalk, K. H., Brandenburger, N. P., and Hol, W. G. J. (1983) Biochemistry 22, 2952-2957). In keeping with recent suggestions that the conformational state of the enzyme is dynamically determined, the discrepancy between Ni(CN)4(2-) affinity as determined by physical methods and that by kinetic methods suggests that Ni(CN)4(2-) may be able to distinguish the conformation of the working enzyme from those of the idle forms.     

Compensation of DNA stabilization and destabilization effects caused by cisplatin is partially disturbed in alkaline medium

Binding of the antitumor compound cisplatin to DNA locally distorts the double helix. These distortions correlate with a decrease in DNA melting temperature (Tm). However, the influence of cisplatin on DNA stability is more complex because it decreases the DNA charge density. In this way, cisplatin increases the melting temperature and partially compensates for the destabilizing influence of structural distortions. The stabilization is stronger at low Na+ ion concentration. Due to this compensation, the total decrease in the DNA melting temperature after cisplatin binding is much lower than the decrease caused by the distortions themselves, especially at low [Na+]. It is shown in this study that, besides Na+ concentration, pH also strongly influences the value of a change in the melting temperature caused by cisplatin. In alkaline medium (pH=10.5-10.8), a fall in the melting temperature caused by platination is enhanced several times with respect to neutral medium. Such a stronger drop in Tm is explained by a decrease in pK values of base pairs caused by lowering the charge density under platination that facilitates proton release. At neutral pH, the proton release is low for both control and platinated DNA and does not influence the melting behavior. Therefore, lowering in the charge density under platination, besides stabilization, gives additional destabilization just in alkaline medium. Destabilization caused by structural distortions due to this pH induced compensation of stabilizing effect is more pronounced. In the presence of carbonate ion, destabilization caused by high pH value is strengthened. As a decrease in DNA charge density, interstrand crosslinking caused by cisplatin also increases the DNA stability due to loss in the entropy of the melted state. However, computer modeling of DNA stability demonstrates that interstrand crosslinks formed by cisplatin do not stabilize long DNA. It is shown that the increase in Tm caused by interstrand crosslinking itself is compensated for by a local destabilization of the double helix at the sites of location of interstrand crosslinks formed by cisplatin.     

Characterization of novel imidazole derivative, JM-8686, a potent inhibitor of allene oxide synthase

The inhibitory properties of a first synthetic jasmonic acid biosynthesis inhibitor, JM-8686, were investigated. Steady-state kinetic analysis indicates that the compound is a competitive inhibitor of allene oxide synthase (AOS) with a K(i) value of approximate 0.62+/-0.15 microM. Dialysis experiment indicates that AOS inactivation by JM-8686 is reversible. The optical difference spectroscopy analysis of JM-8686 and AOS interaction indicates that JM-8686 induced type II binding spectra with a K(d) value of approximate 1.6+/-0.2 microM, suggesting that JM-8686 binds to the prosthetic heme iron of AOS. Comparison of the inhibitory potency of the compound against HPL (CYP74B) from tomato revealed that JM-8686 was a highly selective inhibitor for AOS.     

Nucleotide sequence analysis, overexpression in Escherichia coli and kinetic characterization of Anacystis nidulans catalase-peroxidase

Bifunctional catalase-peroxidases are the least understood type of peroxidases. A high-level expression in Escherichia coli of a fully active recombinant form of a catalase-peroxidase (KatG) from the cyanobacterium Anacystis nidulans (Synechococcus PCC 6301) is reported. Since both physical and kinetic characterization revealed its identity with the wild-type protein, the large quantities of recombinant KatG allowed the examination of both the spectral characteristics and the reactivity of its redox intermediates by using the multi-mixing stopped-flow technique. The homodimeric acidic protein (pI = 4.6) contained high catalase activity (apparent K(m) = 4.8 mM and apparent k(cat) = 8850 s(-1)). Cyanide is shown to be an effective inhibitor of the catalase reaction. The second-order rate constant for cyanide binding to the ferric protein is (6.9 +/- 0.2) x 10(5) M(-1 )s(-1) at pH 7.0 and 15 degrees C and the dissociation constant of the cyanide complex is 17 microM. Because of the overwhelming catalase activity, peroxoacetic acid has been used for compound I formation. The apparent second-order rate constant for formation of compound I from the ferric enzyme and peroxoacetic acid is (1.3 +/- 0.3) x 10(4 )M(-1 )s(-1) at pH 7.0 and 15 degrees C. The spectrum of compound I is characterized by about 40% hypochromicity, a Soret region at 406 nm, and isosbestic points between the native enzyme and compound I at 355 and 428 nm. Rate constants for reduction of KatG compound I by o-dianisidine, pyrogallol, aniline and isoniazid are shown to be (7.3 +/- 0.4) x 10(6) M(-1 )s(-1), (5.4 +/- 0.3) x 10(5) M(-1 )s(-1), (1.6 +/- 0.3) x 10(5) M(-1 )s(-1) and (4.3 +/- 0.2) x 10(4) M(-1 )s(-1), respectively. The redox intermediate formed upon reduction of compound I did not exhibit the classical red-shifted peroxidase compound II spectrum which characterizes the presence of a ferryl oxygen species. Its spectral features indicate that the single oxidizing equivalent in KatG compound II is contained on an amino acid which is not electronically coupled to the heme.     

DNA interaction of dioxycyclobutenedione-(1,2-cyclohexanediamine) platinum(II) complex with potential anticancer activity

Dioxycyclobutenedione-(1,2-cyclohexanediamine)platinum(II), (R,R-DC-Pt) was found to have stronger cytotoxicity against six cancer cell lines than cisplatin and its DNA interactions was studied by calorimetric measurements, (13)C NMR. The binding specificity study of DNA base with R,R-DC-Pt was conducted by HPLC. To understand the molecular mechanism of R,R-DC-Pt with stronger cytotoxicity than that of cisplatin, we studied R,R-DC-Pt interaction with an oligonucleotide, d(ACCACGTGGT)(2), which contained c-H-ras gene encoding GGT by NMR spectroscopy. The oligomer DNA double helix was destroyed almost completely upon the R,R-DC-Pt binding. However under the same condition, the cisplatin binding with DNA was not so affected, and instead another conformation was formed, which suggests that larger damage to DNA can be induced by R,R-DC-Pt complex than that by cisplatin.     

Studies on interaction of an intramolecular charge transfer fluorescence probe: 4'-dimethylamino-2,5-dihydroxychalcone with DNA

The interaction of a new intramolecular charge transfer probe, namely 4'-dimethylamino-2,5-dihydroxychalcone (DMADHC), with calf thymus DNA has been studied. Compared to the spectral characteristics of the free form in aqueous solution, the fluorescence of DMADHC enhanced dramatically accompanying a blueshift of the emission maxima in the presence of DNA. The absorption and fluorescence spectra, salt concentration effect, KI quenching, fluorescence polarization, and DNA denaturation experiments were given. These results give evidence that the DMADHC molecule is inserted into the base-stacking domain of the DNA double helix. The intrinsic binding constant and the binding site number were estimated. The analytical characteristics were also given.     

Kinetics of the reaction of prostaglandin H synthase compound II with ascorbic acid

The reduction of prostaglandin H synthase compound II by ascorbic acid in the presence of diethyldithiocarbamate was studied in 0.1 M phosphate buffer (pH 8.0) at 4.0 +/- 0.5 degrees C, by rapid scan spectrometry and transient state kinetics. A saturation effect and nonzero intercept were observed in the plot of pseudo-first-order rate constant versus ascorbic acid concentration. The saturation behavior suggests formation of a complex between prostaglandin H synthase compound II and ascorbic acid, whereas the nonzero intercept is attributable to the reaction of compound II of prostaglandin H synthase with diethyldithiocarbamate present in the system as a stabilizing agent. A rate equation has been derived which includes all pathways for the conversion of prostaglandin H synthase compound II back to native enzyme. Kinetic parameters for the reduction of compound II by ascorbic acid were obtained. They are the second-order rate constant of (1.4 +/- 0.5) X 10(5) M-1, S-1, for the formation of the compound II-ascorbic acid complex, the first-order rate constant of (14 +/- 4) S-1 for the oxidation-reduction reaction of the complex and its dissociation, and a parameter, Km of 92 +/- 10 microM analogous to the Michaelis-Menten constant. Thus we demonstrate that a quantitative kinetic study on the prostaglandin H synthase reactions can be performed in the presence of diethyldithiocarbamate.     

Investigations of the effects of ethanol on warfarin binding to human serum albumin

Ethanol effects on warfarin binding to human serum albumin (HSA) have been studied by equilibrium dialysis and fluorescence methods at pH 7.4 in phosphate-buffered saline at 37 degrees C. In the presence of various amounts of ethanol fluorescence intensity of bound warfarin decreased significantly but this intensity reduction was not solely from displacement of bound warfarin from HSA. By comparing fluorescence and equilibrium dialysis data we concluded that fluorescence intensity reduction of warfarin was mainly the result of changes in the surrounding environment of the warfarin binding site by ethanol interaction with HSA and that displacement of bound warfarin was not significant compared to the fluorescence intensity changes. The dissociation constant of warfarin binding to HSA decreased with an increasing amount of ethanol. From the changes in fluorescence intensity upon warfarin binding to HSA with the presence of ethanol ranging from 0 to 5.0% the following dissociation constants (Kd) were determined: 0% ethanol 5.39 +/- 0.2 microM, 0.1% ethanol 5.86 +/- 0.1 microM, 0.3% ethanol 5.83 +/- 0.2 microM, 0.5% ethanol 6.76 +/- 0.1 microM, 1% ethanol 7.01 +/- 0.1 microM, 3% ethanol 9.9 +/- 0.7 microM, 5% ethanol 13.01 +/- 0.1 microM. From the equilibrium dialysis with the same ranges of ethanol presence the following Kd values were obtained: 0% ethanol 6. 62 +/- 1.6 microM, 0.1% ethanol 6.81 +/- 1.1 microM, 0.3% ethanol 8. 26 +/- 2.5 microM, 0.5% ethanol 8.86 +/- 1.9 microM, 1% ethanol 11. 01 +/- 4.2 microM, 3% ethanol 20.75 +/- 2.4 microM, 5% ethanol 21.67 +/- 2.2 microM. The results suggest that warfarin bound to HSA was displaced by ethanol. These data indicate that ethanol influence on warfarin binding to HSA may alter the pharmacokinetics of warfarin.     

Potency and property of DNA conformational damage determine ATM activation: two new DNA topological probes

We have shown previously the intercalation geometry of a series of acenaphtho [1,2-b] pyrrole derivatives with DNA double helix in vitro. In this report we chose a couple of intercalating analogues and a Chinese traditional medicine Tanshinone IIA as probes to investigate the response of DNA damage sensor ataxia-telangiectasia mutated (ATM) protein toward the DNA topological change in vivo. The two analogues (1)a (3-(4-Methyl-piperazin)-8-oxo-8H-acenaphtho [1,2-b]pyrrole -9-carbonitrile) and (3)a (3-(3-Dimethylamino-propylamino)-8-oxo-8H-acenaphtho[1,2-b]pyrrole-9- carbonitrile) could unwind double helix to different extents, whereas Tanshinone IIA could wind the double helix. Using a combination of circular dichroism (CD) studies and immunoflurescence assays, we found for the first time that the ATM protein kinase can respond to the unwinding chromatin conformational damage caused by (1)a and (3)a, while it could not be activated by the winding effects caused by Tanshinone IIA. Moreover, the amount of ATM protein phosphorylation is consistent with the degree of unwinding conformational damage. The average number of ATM foci in an MCF-7 cell is 32 +/- 1.5 at 6 microM (1)a, which is significantly higher than the 8 microM (3)a exposure (15 +/- 0.5, p < 0.5). A new couple of DNA topological probes, (1)a and (3)a have been found for the future semi-quantitative investigation of factors involved in the DNA damage pathway.     

Interaction of fluorescence labeled single-stranded DNA with hexameric DNA-helicase RepA: a photon and fluorescence correlation spectroscopy study

Fluorescence correlation spectroscopy (FCS) was used to characterize the interaction of fluorescence labeled single-stranded DNA (ssDNA) with hexameric RepA DNA-helicase (hRepA) encoded by plasmid RSF1010. The apparent dissociation constants, Kd(app), for the equilibrium binding of 12mer, 30mer, and 45mer ssDNA 5'-labeled with BFL to hRepA dimer in the presence of 0.5 mM ATPgammaS at pH 5.8 and 25 degrees C were determined to be 0.58 +/- 0.12, 0.52 +/- 0.07, and 1.66 +/- 0.32 microM, respectively. Binding curves are compatible with one binding site for ssDNA present on hRepA dimer, with no indication of cooperativity. At pH 7.6 in the presence of ATPgammaS and at pH 5.8 in the absence of ATPgammaS, complex formation between ssDNA and hRepA was too weak for measuring complete binding curves by FCS. Under these conditions, the dissociation constant, Kd(app), is in the range between 10 and 250 microM. The kinetics of complex formation at pH 5.8 are faster than the time resolution (approximately 10-20 s) of FCS experiments under pseudo-first-order conditions, with respect to BFL-ssDNA. Photon correlation spectroscopy (PCS) experiments yielded, within the experimental error range, the same values for the apparent hydrodynamic radii, R(h), of hRepA dimer and its complex with ssDNA as determined by FCS (R(h) = 6.6 +/- 1 nm). hRepA starts to aggregate under acidic conditions (相似文献   

Influence of treatment temperature on the genotoxic effects of cisplatin in CHO cells: cytotoxicity, mutagenicity and induction of lesions in DNA

In cells exposed in vitro to the cytotoxic and mutagenic antitumor drug cisplatin (cis-Pt(NH3)2Cl2), various adducts with nuclear DNA are formed. A comparative study was made of the influence of temperature variation during treatment of cultured Chinese hamster ovary (CHO) cells with cisplatin on cytotoxicity, mutation induction and Pt-DNA adduct formation. Before and after treatment (1 h at 32, 37 or 40 degrees C) cells were kept at 37 degrees C. Cytotoxicity increased with temperature; D0 values were 29.6 +/- 1.6, 21.1 +/- 1.2 and 11.4 +/- 0.6 microM at 32, 37 and 40 degrees C, respectively. Pt-DNA binding to DNA at 40 degrees C was 2.0 (+/- 0.3) times as high as at 32 degrees C. This factor remained practically constant over a 24-h post-treatment incubation of the cells, during which about 60% of DNA-bound Pt were removed. As the increase in cytotoxicity between 32 and 40 degrees C was roughly in proportion to that in Pt binding, no substantial changes in the spectrum of adducts appeared to occur. The induction of DNA interstrand cross-links, studied at 32 and 40 degrees C, varied linearly with dose. Influence of temperature on cross-link formation was comparable to that on total Pt binding. Amounts of cross-links highly increased during 24 h after treatment. Plots of cross-links against survival after treatments at 32 and 40 degrees C almost coincided. Induction of 6-thioguanine-resistant (HGPRT) mutants at various cisplatin concentrations did not show a clear temperature dependency. Consequently, equitoxic treatments were significantly more mutagenic at 32 degrees C than at 40 degrees C, the opposite of what has been reported for E. coli.     

Stopped-flow fluorescence studies of HMG-domain protein binding to cisplatin-modified DNA

High-mobility group (HMG) domain proteins bind specifically to the major DNA adducts formed by the anticancer drug cisplatin and can modulate the biological response to this inorganic compound. Stopped-flow fluorescence studies were performed to investigate the kinetics of formation and dissociation of complexes between HMG-domain proteins and a series of 16-mer oligonucleotide probes containing both a 1,2-intrastrand d(GpG) cisplatin cross-link and a fluorescein-modified deoxyuridine residue. Rate constants, activation parameters, and dissociation constants were determined for complexes formed by HMG1 domain A and the platinated DNA probes. The sequence context of the cisplatin adduct modulates the value of the associative rate constant for HMG1 domain A by a factor of 2-4, contributing significantly to differences in binding affinity. The rates of association or dissociation of the protein-DNA complex were similar for a 71 bp platinated DNA analogue. Additional kinetic studies performed with HMG1 domain B, an F37A domain A mutant, and the full-length HMG1 protein highlight differences in the binding properties of the HMG domains. The stopped-flow studies demonstrate the utility of the fluorescein-dU probe in studying protein-DNA complexes. The kinetic data will assist in determining what role these proteins might play in the cisplatin mechanism of action.