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J C Dabrowiak

Publications and source records attributed to J C Dabrowiak.

At least 37 records · Page 2Linked to original sources
PubMed1990

Quantitative footprinting analysis. Binding to a single site.

The theory for measuring ligand binding constants from footprinting autoradiographic data associated with a single binding site is derived. If the ligand and DNA cleavage agent compete for a common site, the spot intensities are not proportional to the amount of DNA not blocked by ligand. The analysis of a single site is experimentally illustrated by using results for the anticancer drug actinomycin D interacting with the duplex d(TAGCGCTA)2 as probed with the hydrolytic enzyme DNase I.

Autoradiography↗
PubMed1990

Antitumor and DNA-binding properties of a group of oligomeric complexes of Pt(II) and Pt(IV).

The antitumor and DNA-binding properties of a group of oligomeric platinum(II) and platinum(IV) complexes are described. The compounds, having the stoichiometry [cis-PtII(X)2(mu-OH)]2(NO3)2, where X is NH3, NH2CH2CH3, and NH2CH(CH3)2, were found to be inactive or only weakly active against L-1210 leukemia. In vitro studies involving PM2-DNA show that these compounds bind to and unwind closed circular DNA in a manner similar to cis-PtII-(NH3)2Cl2. The Pt(IV) complexes produced by hydrogen peroxide oxidation of the Pt(II) dimers are inactive as antitumor agents and are incapable of unwinding PM2-DNA. The cyclotrimer [cis-PtII(RR-DACH)(mu-OH)]3(NO3)3, where RR-DACH is (R,R)-1,2 diaminocyclohexane, exhibits potent antitumor activity against L-1210 leukemia and modest activities with B-16 and M5076 tumor lines. This compound platinates DNA, causing DNA unwinding and mobility shifts.

Animals↗
PubMed1989

Quantitative footprinting analysis using a DNA-cleaving metalloporphyrin complex.

The results of quantitative footprinting studies involving the antiviral agent netropsin and a DNA-cleaving cationic metalloporphyrin complex are presented. An analysis of the footprinting autoradiographic spot intensities using a model previously applied to footprinting studies involving the enzyme DNase I [Ward, B., Rehfuss, R., Goodisman, J., & Dabrowiak, J. C. (1988) Biochemistry 27, 1198-1205] led to very low values for netropsin binding constants on a restriction fragment from pBR-322 DNA. In this work, we show that, because the porphyrin binds with high specificity to DNA, it does not report site loading information in the same manner as does DNase I. We elucidate a model involving binding equilibria for individual sites and include competitive binding of drug and porphyrin for the same site. The free porphyrin and free drug concentrations are determined by binding equilibria with the carrier (calf thymus DNA) which is present in excess and acts as a buffer for both. Given free porphyrin and free netropsin concentrations for each total drug concentration in a series of footprinting experiments, one can calculate autoradiographic spot intensities in terms of the binding constants of netropsin to the various sites on the 139 base pair restriction fragment. The best values of these binding constants are determined by minimizing the sum of the squared differences between calculated and experimental footprinting autoradiographic spot intensities. Although the determined netropsin binding constants are insensitive to the value assumed for the porphyrin binding constant toward its highest affinity sites, the best mean-square deviation between observed and calculated values, D, depends on the choice of (average) drug binding constant to carrier DNA, Kd.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1989

Molecular recognition between oligopeptides and nucleic acids. Sequence specific binding of (4S)-(+)- and (4R)-(-)-dihydrokikumycin B to DNA deduced from 1H NMR, footprinting studies and thermodynamic data.

The sequence specific binding of the antibiotic (4S)-(+)-dihydrokikumycin B and its (4R)-(-) enantiomer, [(S)-1 and (R)-1, respectively] to DNA were characterized by DNase I and MPE footprinting, calorimetry, UV spectroscopy, circular dichroism, and 1H NMR studies. Footprinting analyses showed that both enantiomers [(S)-1 and (R)-1] bind to AT-rich regions of DNA. 1H NMR studies (ligand induced chemical shift changes and NOE differences) of the dihydrkikumycins with d-[CGCAATTGCG]2 show unambiguously that the N to C termini of the ligands are bound to 5'-A5T6T7-3' reading from left to right. From quantitative 1D-NOE studies, the AH2(5)-ligand H7 distance of complex A [(S)-1 plus decamer (which is bound more strongly)] and complex B [(R)-1 and decamer] are estimated to be 3.8 +/- 0.3 A and 4.9 +/- 0.4 A, respectively. This difference in binding properties is reflected in the thermodynamic profiles of the two enantiomeric ligands determined by a combination of spectroscopic and calorimetric techniques. The binding free energies (delta G degrees) of (S)-1 and (R)-1 to poly d(AT).poly d(AT) at 25 degrees C are -31.8 and -29.3 kJ mol-1, respectively while the corresponding binding enthalpies (delta H degrees) are -11.3 and -0.8 kJ mol-1. These data permit the construction of models for the binding of the enantiomeric dihydrokikumycins to DNA and account for the more efficient binding of the natural (S) isomer to DNA.

Anti-Bacterial Agents↗
PubMed1989

Esperamicins, a class of potent antitumor antibiotics: mechanism of action.

The esperamicins represent a class of antitumor antibiotics characterized by an unusual chemical core structure and extremely potent cytotoxicity. The mechanism by which these drugs produce cytotoxicity was investigated and found to be related to the formation of single- and double-strand DNA breaks. Using five structurally related analogs, we defined a structure-activity relationship for cytotoxicity in various eukaryotic and DNA-repair-deficient prokaryotic cell lines, for DNA breakage in a human colon carcinoma cell line, and for DNA breakage in vitro in pBR322 DNA. Mild reducing agents such as dithiothreitol greatly increased the DNA breakage potency of these analogs in vitro. Results suggest that the pendant aromatic chromophore of esperamicin A1 may contribute to the uptake of the drug into cells but may also hinder double-strand DNA break formation. Little DNA breakage specificity was observed for the drug in a 139-base-pair fragment of pBR322 DNA. Evidence supports a previously proposed mechanism whereby esperamicins may produce the observed DNA breaks through reduction of the methyl trisulfide group to a thiolate anion followed by a Michael addition of the anion across the alpha,beta-unsaturated ketone. This addition may result in the saturation of the bridgehead double bond, thus allowing the two triple bonds to approach each other, causing cyclization of the diyn-ene to form a phenylene diradical. It is likely that this diradical is the active form of the drug responsible for single- and double-strand DNA breakage produced by this class of antitumor agents.

Aminoglycosides↗
PubMed1988

Rate enhancements in the DNase I footprinting experiment.

Footprinting experiments for DNase I digests of a 139-base-pair segment of pBR-322 DNA in the presence of either netropsin or actinomycin D were carried out. Plots of oligonucleotide concentration as a function of drug concentration were analyzed to study the enhancement in cleavage rates at approximately 30 sites, accompanying drug binding at other sites. The pattern of enhancements is not consistent with drug-induced DNA structural changes, but agrees with a redistribution mechanism involving DNase I. Since the total number of enzyme molecules per fragment remains unchanged, drug binding at some sites increases the enzyme concentration at other sites, giving rise to increased cleavage. The consequences of the redistribution mechanism for analysis of footprinting experiments are indicated.

Base Composition↗
PubMed1988

Determination of netropsin-DNA binding constants from footprinting data.

A theory for deriving drug-DNA site binding constants from footprinting data is presented. Plots of oligonucleotide concentration, as a function of drug concentration, for various cutting positions on DNA are required. It is assumed that the rate of cleavage at each nucleotide position is proportional to the concentration of enzyme at that nucleotide and to the probability that the nucleotide is not blocked by drug. The probability of a nucleotide position not being blocked is calculated by assuming a conventional binding equilibrium for each binding site with exclusions for overlapping sites. The theory has been used to evaluate individual site binding constants for the antiviral agent netropsin toward a 139 base pair restriction fragment of pBR-322 DNA. Drug binding constants, evaluated from footprinting data in the presence of calf thymus DNA and poly(dGdC) as carrier and in the absence of carrier DNA, were determined by obtaining the best fit between calculated and experimental footprinting data. Although the strong sites on the fragment were all of the type (T.A)4, the value of the binding constant was strongly sequence dependent. Sites containing the dinucleotide sequence 5'-TA-3' were found to have significantly lower binding constants than those without this sequence, suggesting that an adenine-adenine clash produces a DNA structural alteration in the minor groove which discourages netropsin binding to DNA. The errors, scope, and limitations associated with the method are presented and discussed.

Base Composition↗
PubMed1987

Molecular recognition between oligopeptides and nucleic acids. Monocationic imidazole lexitropsins that display enhanced GC sequence dependent DNA binding.

A series of monocationic lexitropsins, or information-reading oligopeptides, were synthesized to minimize and offset the AT bias for doubly cationic ligands bound in the minor groove of DNA. The compounds possess an N-formyl group in place of the guanidinium moiety normally present in netropsin. By systematic replacement of the N-methylpyrrole groups of the dipeptide with N-methylimidazole, a remarkably high degree of sequence specificity was obtained. One of the compounds having two N-methylimidazole residues was found to exhibit dramatically altered specificity when compared with netropsin and preferred to bind to the sequence 5'-CCGT-3' 3'-GGCA-5'. The structural elements underlying sequence recognition in terms of the model for the netropsin-DNA interaction are presented and discussed.

Base Sequence↗
PubMed1987

Comparative antitumor studies on platinum(II) and platinum(IV) complexes containing 1,2-diaminocyclohexane.

The synthesis and characterization of a group of platinum(IV) compounds containing the various isomeric forms of 1,2-diaminocyclohexane (DACH) are described. Antitumor tests with the new complexes, as well as with other platinum(II) compounds containing the DACH ligand, revealed that trans,cis-PtIV(SS-DACH)(OH)2Cl2, 7, is more active than its mirror image, trans,cis-PtIV(RR-DACH)(OH)2Cl2, 6, against L1210 leukemia implanted in mice. However, activity is dependent on the tumor model, and against B16 melanoma implanted in mice, the activities of the two enantiomers are reversed, with 6 being more active than 7. The results of the tests are discussed in light of the mechanism by which Pt(IV) compounds are believed to express their antitumor effects.

Animals↗
PubMed1987

Quantitative footprinting analysis of the netropsin-DNA interaction.

The results of a series of quantitative footprinting experiments of the netropsin-DNA interaction as studied using two different DNA cleaving probes, the enzyme DNase I and a cationic manganese porphyrin complex, are described. Plots of the relative change in oligonucleotide concentration as a function of drug concentration, covering approximately 110 base pairs of a DNA restriction fragment, revealed netropsin induced changes in the cleavage rates of both probes. These appeared as inhibitions for the binding sites, enhancements where no binding took place, and enhancement/inhibitions for the weak binding sites. Determination of the concentration of drug necessary to reduce the amount of a particular oligomer to half of its initial value allowed a ranking of the affinities of the various binding sites on the fragment. In addition to uncovering the location of a number of overlapping netropsin binding sites, the data allowed additional insight on the manner in which both probes alter their DNA cleavage rates in the drug-footprinting experiment.

Antiviral Agents↗
PubMed1986

DNA binding specificity of a series of cationic metalloporphyrin complexes.

The sequence specificities of a series of cationic metalloporphyrins toward a 139 base pair restriction fragment of pBR-322 DNA have been studied by DNase I footprinting methodology. Analysis using controlled digests and quantitative autoradiography/microdensitometry revealed that the 5- and 6-coordinate complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine, MT4MPyP, where M is Mn, Fe, Co, and Zn, were found to bind to AT regions of DNA. Footprinting analysis involving the radiolabel on the opposing strand of restriction fragment showed site skewing in the direction of the 3' end of the fragment, indicating that the porphyrins bind in the minor groove of DNA. The significant increase in DNase I catalyzed hydrolysis observed in various regions of the fragment appeared to be primarily due to a decrease in available substrate DNA upon porphyrin binding with possible contributions from structural changes in DNA caused by ligand binding. The complexes NiT4MPyP and CuT4MPyP were found to bind to both AT and GC regions of the fragment, producing different degrees of inhibition in the two regions. Since the outside-binding porphyrins can neither intercalate or effectively hydrogen bond to DNA, they appear to read sequence by responding to steric and/or electrostatic potential effects located in the minor groove of DNA.

Base Sequence↗
PubMed1986

Cationic porphyrins as probes of DNA structure.

The DNA binding specificity of a group of cationic manganese porphyrin complexes has been examined using DNase I footprinting methodology and by observing the sites of porphyrin-induced DNA strand scission in the presence of potassium superoxide. The compounds, which possess systematic changes in total charge, its distribution on the periphery on the macrocycle and ligand shape, bind in the minor groove of AT rich regions of DNA. While changes in total charge and charge arrangement do not significantly influence specificity, a shape change which blocks close ligand contact with the minor groove relaxes the original AT specificity causing the compound to cleave at both AT and GC sites. The observed changes in binding sequence specificity were interpreted in terms of electrostatic and steric factors associated with both the compounds and DNA.

Base Sequence↗
PubMed1986

Molecular recognition between oligopeptides and nucleic acids: novel imidazole-containing oligopeptides related to netropsin that exhibit altered DNA sequence specificity.

Oligopeptides have been synthesized that are structurally related to the antiviral antitumor antibiotic netropsin, but in which each of the pyrrole units is successively replaced by an imidazole moiety, as well as their di- and triimidazole-containing counterparts. These compounds bind to duplex DNA with constants in the range (1.06-1.98) X 10(6) M-1 but not to single-stranded DNA. Since they bind to T4 DNA, it is inferred that, like the parent antibiotic netropsin, they are also minor groove selective. This series of compounds exhibits a progressively decreasing preference for AT sites in binding studies with both native DNAs and synthetic oligonucleotides and a corresponding increasing acceptance of GC base pairs. Footprinting experiments utilizing a 139 base pair HindIII/NciI restriction fragment from pBR 322 DNA revealed that these lexitropsins, or information-reading oligopeptides, recognize more sites than the parent netropsin. In addition, some regions of enhanced nuclease action as the result of drug binding to the fragment were identified. The diimidazole compound in particular recognizes GC-rich sites, implying the formation of new hydrogen bonds between G-C(2)NH2 in the minor groove and the additional N3 imidazole nitrogens. It is clear however that, since the lexitropsins appear to tolerate the original (AT)4 site, an N-methylimidazole group on the ligand will permit either a GC or AT base pair in the binding sequence. Another factor that may be significant in molecular recognition is the high negative electrostatic potential of A X T regions of the minor groove, which is likely to strongly influence binding of these cationic species to DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1986

DNA cleavage specificity of a group of cationic metalloporphyrins.

The ability of a group of water-soluble metalloporphyrins to cleave DNA has been investigated. Incubation of Mn3+, Fe3+, or Co3+ complexes of meso-tetrakis(N-methyl-4-pyridiniumyl)porphine (H2T4MPyP) with DNA in the presence of ascorbate, superoxide ion, or iodosobenzene results in DNA breakage. Comparisons between the rates of porphyrin autodestruction with the rates of strand scission of covalently closed circular PM2 DNA indicate that the porphyrins remain intact during the cleavage process. Analysis of the porphyrin-mediated strand scissions on a 139-base-pair restriction fragment of pBR322 DNA using gel electrophoresis/autoradiography/microdensitometry reveals that the minimum porphyrin cleavage site is (A X T)3. The cleavage pattern within a given site was found to be asymmetric, indicating that porphyrin binding and the strand scission process are highly directional in nature. In addition to an analysis of the mechanism of porphyrin-mediated strand breakage in terms of the DNA cleavage mechanism of methidium-propyl-iron-EDTA and Fe-bleomycin, the potential of the cationic metalloporphyrins as footprinting probes and as new "reporter ligands" for DNA is presented and discussed.

Base Sequence↗
PubMed1986

Deoxyribonucleic acid cleavage specificity of a series of acridine- and acodazole-iron porphyrins as functional bleomycin models.

A series of metalloporphyrins linked through basic chains to certain DNA interactive groups has been synthesized. Several of these agents reproduce the characteristic properties of the antitumor glycopeptide bleomycin, including the oxygen-mediated scission of DNA in the presence of thiols, antibiobic activity under aerobic conditions, and activity against human and animal tumor models. Initial screening by scission of PM2-CCC-DNA identified six of the compounds, including those bearing acridine and acodazole intercalating groups, as the most active. The specificity of the oxygen-mediated scission of a 139 base pair HindIII/NciI restriction fragment of pBR322 by these six selected agents was then determined and compared with the action of pancreatic DNase by densitometric scans. All six of these compounds produce uniform base and sequence neutral cleavage of the restriction fragment at each base site. The six active compounds bear either of two types of intercalators, 6-chloro-2-methoxyacridine or acodazole, and with linkages to the ferric binding domain of -NH(CH2)2-, -NH(CH2)3-, -NH(CH2)4-, or -NH(CH2)3NH(CH2)3- and either porphyrin or deuteroporphyrin moieties. Comparison of the Kassoc values for binding to calf thymus DNA suggests that the enhanced binding observed with the linker -NH(CH2)3NH(CH2)3- contributes to the efficiency of sequence neutral DNA scission and may be a factor in the relative anticancer activities of these agents. The iron porphyrins give no evidence of the production of base propenals in DNA degradation, and the autoradiograms clearly indicate that a phosphate group is attached to the 5' end of the oligomer. The scission is partially suppressible by catalase and superoxide dismutase.(ABSTRACT TRUNCATED AT 250 WORDS)

Acridines↗
PubMed1986

Computer assisted microdensitometric analysis of footprinting autoradiographic DATA.

A system comprised of a linear scanning microdensitometer interfaced to a personal computer has been developed to facilitate analysis of ligand-DNA footprinting autoradiograms. The system, which can be used to record density and sequence information from autoradiographic films, enables the user to relate the area under an autoradiographic band to the concentration of radiolabeled molecules present in the electrophoresis gel. This report describes the computer program which performs the calculations and discusses the ability of the system to accurately determine oligonucleotide concentration, as a function of band separation, photographic response, and the computational algorithm used to calculate band areas.

Autoradiography↗