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P B Hopkins

Publications and source records attributed to P B Hopkins.

At least 19 recordsLinked to original sources
PubMed1998

Effect of nucleosome structure on DNA interstrand cross-linking reactions.

Antitumor agents of the nitrogen mustard family and mitomycin C form interstrand cross-links in duplex DNA. To provide information about the cellular mechanism by which these compounds exert their cytotoxic effects, we examined cross-linking of a nucleosomal core particle formed on a fragment of the 5S RNA gene of Xenopus borealis. For the mustards mechlorethamine, chlorambucil, and melphalan, both sites of monoalkylation and interstrand cross-linking were similar in nucleosomal and free DNA. Some small (two- to three- fold) differences in intensity of cross-linking at some sites were apparent. However, these differences did not appear to correlate with rotational or translational positioning. For mitomycin C, cross-linking was inhibited five- to ten-fold at the nucleosomal dyad and showed attenuation of inhibition toward the ends. Furthermore, rotational positioning also appeared to be a factor, with sites facing inward in the nucleosome less accessible for mitomycin cross-linking. None of these agents demonstrated the 10-base pair periodicity exhibited by hydroxyl radical cleavage of nucleosomal DNA.

Animals↗
PubMed1996

An NMR study of [d(CGCGAATTCGCG)]2 containing an interstrand cross-link derived from a distamycin-pyrrole conjugate.

Minor groove binding compounds related to distamycin A bind DNA with high sequence selectivity, recognizing sites which contain various combinations of A.T and G.C base pairs. These molecules have the potential to deliver cross-linking agents to the minor groove of a target DNA sequence. We have studied the covalent DNA-DNA cross-linked complex of 2,3- bis(hydroxymethyl)pyrrole-distamycin and [d(CGCGAATTCGCG)]2. The alkylating pyrrole design is based on the pharmacophore of mitomycin C and is similar in substructure to another important class of natural products, the oxidatively activated pyrrolizidine alkaloids. Ligand-DNA NOEs confirm that the tri(pyrrole-carboxamide) unit of the ligand is bound in the minor groove of the central A+T tract. Unexpectedly, it is shifted by 1 bp with respect to the distamycin A binding site on this DNA sequence. The cross-link bridges the 2-amino position of two guanine residues, G4 and G22. The C3.G22 and G4.C21 base pairs exhibit Watson-Crick base pairing, with some local distortion, as evidenced by unusual intensities observed for DNA-DNA NOE cross-peaks. The model is compared with a related structure of a cross-linked mitomycin C:DNA complex.

Base Sequence↗
PubMed1996

Bending of DNA by the mitomycin C-induced, GpG intrastrand cross-link.

Mitomycin C (MC) forms interstrand and intrastrand cross-link adducts and monoalkylation products (monoadducts) with DNA. Each of the three types of adducts was incorporated site-specifically into both a 15-mer and a 21-mer oligodeoxyribonucleotide duplex. The adduct-containing duplexes were 32P-phosphorylated and ligated to form multimers, which were then analyzed for anomalous electrophoretic mobility by nondenaturing polyacrylamide gel electrophoresis, using the method of Koo and Crothers [(1988) Proc. Natl. Acad. Sci. U.S.A. 85, 1763-1767] in order to detect DNA curvature caused by the adducts. The intrastrand cross-link adduct was found to induce a 14.6 +/- 2.0 degrees DNA bend per lesion (minimum value) while no DNA bending was detected for either the interstrand cross-link or the monoadduct. Molecular mechanics modeling indicated that the possible origin of the bend lies in a considerable deviation from parallel of the normals to the best planes of the intrastrand cross-linked guanines, due to a shorter than normal distance between their N2 atoms forced upon them by the cross-link. The observed bending by the MC intrastrand lesion may be the cause of the increased flexibility of MC-modified DNA, localized to distinct regions, as observed in earlier work by hydrodynamic methods and electron microscopy. The MC adduct-caused DNA bend may serve as a recognition site for certain DNA-binding proteins.

Cross-Linking Reagents↗
PubMed1995

Solution structure of a cisplatin-induced DNA interstrand cross-link.

The widely used antitumor drug cis-diamminedichloroplatinum(II) (cisplatin or cis-DDP) reacts with DNA, cross-linking two purine residues through the N7 atoms, which reside in the major groove in B-form DNA. The solution structure of the short duplex [d(CAT-AGCTATG)]2 cross-linked at the GC:GC site was determined by nuclear magnetic resonance (NMR). The deoxyguanosine-bridging cis-diammineplatinum(II) lies in the minor groove, and the complementary deoxycytidines are extrahelical. The double helix is locally reversed to a left-handed form, and the helix is unwound and bent toward the minor groove. These findings were independently confirmed by results from a phase-sensitive gel electrophoresis bending assay. The NMR structure differs markedly from previously proposed models but accounts for the chemical reactivity, the unwinding, and the bending of cis-DDP interstrand cross-linked DNA and may be important in the formation and repair of these cross-links in chromatin.

Antineoplastic Agents↗
PubMed1995

Monitoring DNA dynamics using spin-labels with different independent mobilities.

The electron paramagnetic resonance (EPR) spectra of spin-labeled DNA duplexes, both bound to DEAE-Sephadex and free in solution, have been analyzed. The nitroxide spin-labels are covalently linked to a deoxyuridine residue using either a monoacetylene or diacetylene tether. This difference in tether length produces a dramatic difference in the independent mobility of the nitroxide relative to the DNA. In the case of the monoacetylene tether, the motion of the nitroxide has previously been shown to be tightly coupled to that of the DNA duplex. With the diacetylene tether, there is considerable independent motion of the probe. The diacetylene tether is intended to minimize the possibility of the nitroxide producing a perturbation of the dynamics of DNA. It is demonstrated here that, when coupled via the diacetylene tether, the nitroxide undergoes a rapid uniaxial rotation about the tether. A detailed analysis of the EPR spectrum of duplex DNA in solution, spin-labeled using the diacetylene tether, demonstrates that the motion of the nitroxide can be modeled in terms of this independent uniaxial rotation together with motion of the DNA which is consistent with the global tumbling of the duplex. As was previously found using the monoacetylene tether, there is no evidence of rapid, large-amplitude motions of the base pair in the EPR spectrum of a nitroxide coupled to duplex DNA via the diacetylene tether. This result confirms the small amplitudes of internal motion, local and collective, previously observed in duplex DNA with the monoacetylene-tethered nitroxide.

Acetylene↗
PubMed1995

A mechlorethamine-induced DNA interstrand cross-link bends duplex DNA.

The dG-to-dG, DNA-DNA interstrand cross-link at the duplex sequence 5'-d(GNC) formed by the antitumor drug mechlorethamine (bis(2-chloroethyl)methylamine) was studied both theoretically and experimentally. Computer models of cross-linked DNA were energy minimized using molecular mechanics. The energy minimized structures possessed local distortion of the DNA helix, especially propeller twisting and buckling, caused by the tether length being too small to bridge the spacing of N7 atoms of dG at the sequence 5'-d(GNC) in B DNA. Overwinding of 2-6 degrees was present at each of the two dinucleotide steps spanned by the cross-link. The predicted structural changes were compatible with the possibility that this cross-link would introduce a static bend into the DNA double helix axis. An experimental study provided evidence for this induced bending of the helix axis in interstrand cross-linked samples. DNAs containing multiple mechlorethamine-induced interstrand cross-links exhibited anomalously low electrophoretic mobility in polyacrylamide gels when the lesions were separated by one or two turns. From the degree of gel retardation, the cross-linked DNAs were estimated to be bent by 12.4-16.8 degrees per lesion; estimation of the extent to which this bend was induced by the lesion was complicated by a preexisting bend in the non-cross-linked DNAs used. The data did not allow distinction of a static from an anisotropic dynamic bend; "universal" and "hinge" joints were excluded. Anomalous mobility was maximal when the lesion spacing was 21 bp, suggesting a helical repeat of 10.5 bp per turn.

Base Sequence↗
PubMed1995

Ovothiols.

Explore the source record for details and available documents.

Animals↗
PubMed1995

DNA-DNA interstrand cross-linking by cis-diamminedichloroplatinum(II): N7(dG)-to-N7(dG) cross-linking at 5'-d(GC) in synthetic oligonucleotides.

The nucleotide sequence specificity of the DNA-DNA interstrand cross-linking reaction of cis-diamminedichloroplatinum(II) (cis-DDP) was studied in synthetic oligonucleotides. Of six self-complementary DNAs tested, only those containing the central sequence 5'-d(GC) formed appreciable interstrand cross-linked product, as assayed by denaturing polyacrylamide gel electrophoresis (DPAGE). The nucleotide connectivity of the interstrand cross-link was defined by sequence random oxidative fragmentation followed by DPAGE, revealing that dG residues on opposite strands at the sequence 5'-d(GC) were connected to one another. The covalent structure of the cross-link was established following hydrolysis of the phosphodiester backbone of a structurally homogeneous sample of a cis-DDP interstrand cross-linked DNA tetradecamer. HPLC analysis of the hydrolysate returned all of the deoxynucleoside residues from the starting DNA except for two deoxyguanosine residues. Also returned was diammine-bis-[N7-(2'- deoxyguanosyl)]platinum(II)2+, identified by a combination of spectroscopic methods, and comparison to a synthetic authentic sample. This study directly establishes that cis-DDP forms interstrand cross-links at the duplex sequence 5'-d(GC), linking deoxyguanosine residues on opposite strands at N7 through a bridging platinum atom. Computer simulation of the interstrand cross-linked product using molecular mechanics energy minimization and molecular dynamics revealed significant structural reorganization at the site of the cross-link including a ca 40 degrees angle between the platinated guaninyl residues, which propagated to adjacent residues by base stacking to yield duplexes bent by some 30 degrees toward the major groove.

Antineoplastic Agents↗
PubMed1993

Motions of short DNA duplexes: an analysis of DNA dynamics using an EPR-active probe.

The dynamics of a series of four DNA duplexes of length 12, 24, 48, and 96 base pairs have been studied using an electron paramagnetic resonance (EPR) active nitroxide spin-label covalently attached to a thymidine located near the center of each duplex. The linear EPR spectra were simulated by solving the stochastic Liouville equation for anisotropic rotational diffusion. The diffusion tensor for global rotation of the duplex was predicted from hydrodynamic theory for a right circular cylinder. All internal motions, assumed to be rapid, are modeled by reduced electron Zeeman and hyperfine tensor anisotropies. Best fit simulations to the data were then obtained by adjusting the total amplitude of all internal dynamics. The local, length-independent and the collective, length-dependent contributions to the internal dynamics were separated by determining the total amplitude of internal motion as a function of duplex length. The major axis of the spin tensors was determined to be tilted 20 degrees from the helix axis. As a result, the spin-label is most sensitive to flexural motions of the DNA duplex. It is found that the global tumbling of duplex is accurately modeled by hydrodynamic theory. The length-independent motion is characterized by a root-mean-squared amplitude of oscillation of 10 degrees in two dimensions at 20 degrees C and has a strong temperature dependence, indicating that the local structure of the DNA changes with temperature. The length dependence of the internal dynamics leads to an estimate of the dynamic flexural persistence length of 2500 +/- 340 A. There was no statistically significant difference between models assuming a harmonic or a square-well local bending potential.

Base Sequence↗
PubMed1993

Site-specific metal-induced damage of mitomycin C-crosslinked DNA fragments in the presence of sodium dithionite.

Purified singly mitomycin C-crosslinked DNA duplex fragments were treated with Cu(II) in the presence of sodium dithionite. Cleavage products were analyzed through single-nucleotide resolving denaturing polyacrylamide gel electrophoresis. In addition to the previously described non-specific cleavage, which may be attributed to the production of hydroxyl radical, a novel site-specific cleavage in the immediate vicinity of the MC-crosslink was observed. Possible mechanisms of this reaction are discussed.

Base Sequence↗
PubMed1991

Sequence preferences of DNA interstrand crosslinking agents: quantitation of interstrand crosslink locations in DNA duplex fragments containing multiple crosslinkable sites.

A general approach to the quantitative study of the sequence specificity of DNA interstrand crosslinking agents in synthetic duplex DNA fragments is described. In the first step, a DNA fragment previously treated with an interstrand crosslinking agent is subjected to denaturing PAGE. Not only does this distinguish crosslinked from native or monoadducted DNA, it is shown herein that isomeric crosslinked DNAs differing in position of the crosslink can in some cases be separated. In the second stage, the now fractionated crosslinked DNAs isolated from denaturing PAGE are subjected to fragmentation using iron(II)/EDTA. For those fractions which are structurally homogeneous, analysis of the resulting fragment distribution has previously been shown to reveal the crosslink position at nucleotide resolution. It is shown herein that in fractions which are structurally heterogeneous due to differences in position of crosslink, this analysis quantifies the relative extent of crosslinking at distinct sites. Using this method it is shown that reductively activated mitomycin C crosslinks the duplex sequences 5'-GCGC and 5'-TCGA with 3 +/- 1:1 relative efficiency.

Base Sequence↗
PubMed1990

Sequence preferences of DNA interstrand cross-linking agents: dG-to-dG cross-linking at 5'-CG by structurally simplified analogues of mitomycin C.

The nucleotide sequence preferences of the DNA interstrand cross-linking agents dehydroretronecine diacetate (DHRA), 2,3-bis(acetoxymethyl)-1-methylpyrrole (BAMP), dehydromonocrotaline, and dehydroretrorsine were studied by using synthetic DNA duplex fragments and polyacrylamide gel electrophoresis (PAGE). These agents have structural features in common with the reductively activated aziridinomitosene of mitomycin C (MC). Like MC, they preferentially cross-linked DNA duplexes containing the duplex sequence 5'-CG. For DHRA and BAMP interstrand cross-linked DNA duplexes, PAGE analysis of iron(II)-EDTA fragmentation reactions revealed the interstrand cross-links to be deoxyguanosine to deoxyguanosine (dG-to-dG), again analogous to DNA cross-links caused by MC. Unlike MC, DHRA could be shown to dG-to-dG cross-link a 5'-GC sequence. Furthermore, the impact of flanking sequence on the efficiency of interstrand cross-linking at 5'-CG was reduced for BAMP, with 5'-TCGA and 5'-GCGC being equally efficiently cross-linked. Possible origins of the 5'-CG sequence recognition common to all of the agents are discussed. A model is presented in which the transition state for the conversion of monoadducts to cross-links more closely resembles ground-state DNA at 5'-CG sequences.

Base Sequence↗
PubMed1990

Ovothiols as free-radical scavengers and the mechanism of ovothiol-promoted NAD(P)H-O2 oxidoreductase activity.

Racemic ovothiol A [(+/-)-1a] and the ovothiol model compound 1,5-dimethyl-4-mercaptoimidazole (DMI, 2) were found to scavange the free radicals Fremy's salt (4) and Banfield' radical (5) much more rapidly than did the thiol antioxidant glutathione. Ovothiol A also scavenges the tyrosyl radical, with efficiency comparable to that of ascorbic acid and the tocopherol analogue trolox (3). The ovothiol model compound DMI was found to scavenge superoxide with a rate constant comparable to that of the reaction between superoxide and glutathione. These results suggest both a free-radical scavenging role for the ovothiols and a mechanism by which the ovothiols confer NAD(P)H-O2 oxidoreductase activity upon the enzyme ovoperoxidase. Investigation of this mechanism implicates the ovothiol thiyl radical and the NAD radical as key intermediates. The ovothiyl radical appears to be unreactive toward oxygen but highly reactive toward NADH. An estimate of the one-electron oxidation potential of the ovothiol anion is presented. The physical basis for the stability of the ovothiol free radical is discussed.

Amino Acids, Sulfur↗
PubMed1989

Sequence- and structure-dependent DNA base dynamics: synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA.

A nitroxide spin-labeled analogue of thymidine (1a), in which the methyl group is replaced by an acetylene-tethered nitroxide, was evaluated as a probe for structural and dynamics studies of sequence specifically spin-labeled DNA. Residue 1a was incorporated into synthetic deoxyoligonucleotides by using automated phosphite triester methods. 1H NMR, CD, and thermal denaturation studies indicate that 1a (T*) does not significantly alter the structure of 5'-d(CGCGAATT*CGCG) from that of the native dodecamer. EPR studies on monomer, single-stranded, and duplexed DNA show that 1a readily distinguishes environments of different rigidity. Comparison of the general line-shape features of the observed EPR spectra of several small duplexes (12-mer, 24-mer) with simulated EPR spectra assuming isotropic motion suggests that probe 1a monitors global tumbling of small duplexes. Increasing the length of the DNA oligomers results in significant deviation from isotropic motion, with line-shape features similar to those of calculated spectra of objects with isotropic rotational correlation times of 20-100 ns. EPR spectra of a spin-labeled GT mismatch and a T bulge in long DNAs are distinct from those of spin-labeled Watson-Crick paired DNAs, further demonstrating the value of EPR as a tool in the evaluation of local dynamic and structural features in macromolecules.

Base Sequence↗
PubMed1989

A qualitative fluorescence-based assay for tyrosyl radical scavenging activity: ovothiol A is an efficient scavenger.

A method for determining relative tyrosyl radical scavenging activity of antioxidants which requires only a standard fluorometer and commercially available materials is presented. Ultraviolet irradiation of aqueous tyrosine solutions containing superoxide dismutase and catalase produces fluorescent dityrosine residues via dimerization of photogenerated tyrosyl radicals. Added antioxidants suppress the buildup of fluorescence by scavenging the tyrosyl radicals. A correlation exists between the ability of a substance to suppress dityrosine formation and the substance's one-electron oxidation potential. This method demonstrates that ovothiol A scavenges tyrosyl radicals much more efficiently than glutathione or cysteine, resembling instead the known biological radical scavengers uric acid and ascorbic acid and the alpha-tocopherol analog trolox.

Animals↗
PubMed1987

Fluoro ketone containing peptides as inhibitors of human renin.

The pentapeptide BOC-Phe-Phe-difluorostatone-Leu-Phe-NH2 has been prepared and found to be a potent inhibitor of human renin. This compound contains a difluoromethylene ketone group that exists predominantly in the hydrated form in water. The difluorostatone-containing peptide is 7-fold and 22-fold more potent than the analogous statine- and statone-containing peptides, respectively. Structure/activity analysis of the most potent inhibitor was carried out by replacing some of the peptide bonds with trans-alkenes. In all cases, a dramatic loss in binding to renin was observed. A number of statine-containing inhibitors of renin have been reported and this work suggests that the replacement of statine with difluorostatone will yield more potent compounds.

Amino Acid Sequence↗
PubMed1987

Probes for double helical DNA sequence information: molecular mechanics study of a proposed model.

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.

Base Composition↗