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S A Winkle

Publications and source records attributed to S A Winkle.

17 recordsLinked to original sources
PubMed1992

Using lambda exonuclease inhibition assays to map carcinogen binding sites.

Previous restriction mapping studies (M.A. Mallamaci, D.P. Reed and S.A. Winkle, J. Biomolecular Structure and Dynamics, in press (1992)) have indicated that a small number of locations on the plasmid pBR322 may be high affinity binding sites for the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF). PBR322 was reacted with acetoxyAAF to produce DNA with one, three or seven acetoxyAAF moieties per DNA molecule. Thus only the higher affinity binding sites are affected. Subsequent digestion with the restriction enzyme Hinf I produced fragments containing previously indicated locations of potential acetoxyAAF binding sites. Fragments thought not to contain binding sites were also examined as controls. The isolated fragments, singly 32P end-labeled, were digested with lambda exonuclease. The three fragments suspected of containing acetoxyAAF binding sites possess new lambda exonuclease inhibition sites when the fragments are obtained from acetoxyAAF reacted DNA. No such inhibition sites are found with the two fragments suggested previously not to contain acetoxyAAF binding sites. These carcinogen produced inhibition sites occur in sequences which are similar, suggesting that acetoxyAAF preferentially may target a small number of sequences.

Acetoxyacetylaminofluorene↗
PubMed1992

Restriction enzyme mapping of carcinogen binding regions on pBR322.

Previous equilibrium binding experiments (S.A. Winkle and T.R. Krugh, Nucleic Acids Res. 9, 3175-3186 (1981)) suggested that the carcinogen N-hydroxy-N-acetyl-2-aminofluorene might exhibit preferential binding to a small number of sites on phiX174 DNA. To examine whether the covalently binding analogue N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF) also possesses high affinity sites, the plasmid pBR322 was reacted with 3H labeled acetoxyAAF to give one to sixteen adducts per DNA molecule. Thus only higher affinity sites would be affected. The DNA was subsequently cleaved with either Alu I, Hae III, Hha I, Hinf I or Hpa II restriction endonuclease and the restriction fragments isolated by gel electrophoresis. Examination of the distribution of 3H acetoxyAAF among the fragments was not random but, rather, with each enzyme, the acetoxyAAF was found predominantly in a few fragments. The locations of the bands containing the acetoxyAAF for each enzyme overlap--suggesting that there are regions on pBR 322 which contain high affinity sites for acetoxyAAF binding.

Acetoxyacetylaminofluorene↗
PubMed1992

Locating binding sites for the carcinogen N-acetoxy-N-acetyl-2-aminofluorene using restriction enzyme inhibition assays.

Restriction enzyme inhibition studies have been employed to map the locations of high affinity binding sites of the carcinogen N-acetoxy-N-acetyl-2-aminofluorene (acetoxyAAF) on pBR322, phiX174 and SV40 DNAs. Bound carcinogen levels were kept low (less than 20 bound AAF moieties per DNA molecule) in order to observe only the binding to the high affinity sites. Inhibition of certain restriction enzymes was observed in a limited number of locations on these DNAs. Inhibition increased as bound AAF increased and the particular restriction enzymes inhibited varied with location. On all three DNAs, activities of these enzymes was not affected in other locations. Comparison of the sequences at the sites of inhibition on the three DNAs indicates that all sites have common sequence elements: the presence of either the sequence T(C/G)TT(G/C) or the sequence T(G/C)CTT(G/C).

Acetoxyacetylaminofluorene↗
PubMed1992

The interface between an alternating CG motif and a random sequence motif displays altered nuclease activity.

Previously we described the B-Z junctions produced in oligomers containing (5meCG)4 segments in the presence of 5.0 M NaCl or 50 uM Co(NH3)6+3 [Sheardy, R.D. & Winkle, S.A., Biochemistry 28, 720-725 (1989); Winkle, S.A., Aloyo, M.C., Morales, N., Zambrano, T.Y. & Sheardy, R.D., Biochemistry 30, 10601-10606 (1991)]. The circular dichroism spectra of an analogous unmethylated oligomer containing (CG)4, termed BZ-IV, in 5.0 M NaCl and in 50 uM Co(NH3)+3 suggest, however, that this oligomer does not form a B-Z hybrid. BZ-IV possesses Hha I sites (CGCG) in the (CG)4 segment and an Mbo I site (GATC) at the terminus of the (CG)4 segment. BZ-IV is equally digestible in the presence and absence of cobalt hexamine by Hha I, further indicating that the structure of BZ-IV is fully B-like under these conditions. The Mbo I cleavage site at the juncture between the (CG)4 segment and the adjacent random segment displays enhanced cleavage by both Mbo I and its isoschizomer Sau3AI in the presence of cobalt hexamine. In addition, exonuclease III digestion of BZ-IV is inhibited at this juncture. Actinomycin inhibits Mbo I activity in the presence of cobalt hexamine but not in the absence. Together, these results suggest that enzymes recognize the interfaces of (CG)n and adjacent random sequences as altered substrates even in the absence of a B-Z junction formation.

Base Sequence↗
PubMed1991

Enhanced reactivity of a B-Z junction for cleavage by the restriction enzyme MboI.

We have been investigating the structure, dynamics, and ligand-binding properties of the interface that exists between a right-handed conformation and a left-handed conformation (i.e., a B-Z junction) in synthetic DNA oligomers. Since exo- and endonuclease activity is known to be sensitive to the conformation of the template DNA, we have designed and synthesized a DNA oligonucleotide of 20 base pairs (designated as BZ-III) with an MboI recognition site (GATC) at the location of a potential B-Z junction. The activity of the MboI enzyme toward this molecule and DNA oligomers that contain multiple MboI sites located at B-Z junctions was monitored in the absence and presence of the Z-conformation-inducing reagent cobalt hexaammine. In all cases, the activity of the enzyme was enhanced in the presence of cobalt hexaammine. The activity of MboI toward BZ-III, in the presence and absence of cobalt hexaammine, was also examined when the DNA oligomer is also in the presence of the DNA binding drugs actinomycin D, ametantrone, or ethidium bromide. In all cases, the activity of the enzyme was inhibited in the presence of drug. The results suggest that B-Z junctions are structurally unique and that this uniqueness may alter nuclease activity at sites in or near the junction.

Base Sequence↗
PubMed1990

Anomalous gel migration of DNA oligomers containing multiple conformational junctions.

We have previously shown that a short 16 base pair DNA oligomer can accommodate a B-Z conformational junction [Sheardy, R. D., & Winkle, S. A. (1989) Biochemistry 28, 720-725]. Results from 1H NMR studies indicated that only three base pairs were involved in the junction and that one of these base pairs was highly distorted. Being interested in the nature of this distortion, we constructed DNA oligomers which have the potential to contain multiple B-Z junctions for polyacrylamide electrophoretic studies. We report that the mobilities displayed by these molecules through acrylamide gels in the absence and presence of cobalt suggest that these molecules run shorter than they actually are. This anomalous migration may be due to structural/dynamic properties of the DNA helix manifested by the periodic distortions of the potential B-Z junctions.

Base Sequence↗
PubMed1989

Temperature-dependent CD and NMR studies on a synthetic oligonucleotide containing a B-Z junction at high salt.

It is now accepted that two or more conformations may exist within the same DNA molecule, thereby generating conformational junctions. The presence of B-Z junctions between right- and left-handed DNA conformations has been detected in plasmids by a number of techniques. Preliminary characterization of the first example of a B-Z junction is a short DNA oligonucleotide has recently been reported [Sheardy, R. D. (1988) Nucleic Acids Res. 16, 1153-1167]. We report additional CD and NMR data that support the existence of the junction in this model oligomer. These studies indicate that only three base pairs are involved in the junction and only one of these is dramatically distorted. Furthermore, the NMR saturation-transfer experiments suggest the junction's internal motion is temperature dependent.

Circular Dichroism↗
PubMed1988

Equilibrium binding of spermine and histamine to salmon sperm DNA and poly(dGdC).

Studies using the solute-enhanced phase partition technique demonstrate that the endogenous amines histamine and spermine bind to both salmon sperm DNA and poly(dGdC) in a markedly cooperative fashion. Both compounds exhibited DNA sequence-dependence effects in their mode of binding. The linear aliphatic spermine molecule binds more strongly than histamine to both DNA types.

Animals↗
PubMed1986

Mixed-mode hydrophobic ion exchange for the separation of oligonucleotides and DNA fragments using HPLC.

Hydrophobic anion exchangers were formed by cobonding both ionic and hydrophobic ligands to silica gel. These phases were used to separate single-stranded oligonucleotides and double-stranded DNA restriction fragments. By varying the ratio of n-octyldimethylsilane and either 3-chloropropyldimethylsilane or 4-chlorobutyldimethylsilane added during silanization a series of mixed-ligand or mixed-mode stationary phases was created. Concentration and ratio of bonded ligands were determined using a new gas chromatography fluorination method. Total ligand coverage was found to approach 2.1 ligands nm-2 for n-octyldimethylsilane. Bonding reproducibility for mixed-mode phases was good. Nucleic acid separations were achieved under gentle mobile phase conditions by using the stationary phase as an easily modifiable variable.

Chromatography, Gas↗
PubMed1986

13C-NMR of ribosyl ApApA, ApApG and ApUpG.

The chemical shifts as well as the 13C-31P coupling constants of the carbon-13 nuclei in single-stranded ApApA, ApApG, and ApUpG are sensitive to sequence and temperature. ApApA and ApApG have similar properties with large shielding (up to 1.7 ppm) of many of the base carbons upon decreasing the temperature from 70 degrees C to 11 degrees C; the base carbons have smaller shielding changes in ApUpG. Large shielding and deshielding effects are observed for the 1', 3', 4' and 5'-carbons over this temperature range. Analysis of the 13C-31P couplings measured at the 4' ribose carbons show that the population of the anti rotamer about O5'-C5' varies from 98 to 75%, and is higher in ApApA and ApApG than in ApUpG. The CCOP coupling data at 2' and 4' is consistent with a blend of the -antiperiplanar/-synclinal nonclassical rotamers about the C3'-O3' bond, varying from 89/11% in ApApG to 55/45% in ApUpG. The coupling and chemical shift data support the thesis that ApUpG is stacked much less than the other two molecules. The stacked forms of all three trinucleotides is most easily interpreted by a standard A-RNA model. It is not necessary to invoke the "bulged base" hypothesis [Lee, C.-H. and Tinoco, Jr., I. (1981) Biophysical Chemistry 1, 283-294; Lankhorst, P.P., Wille, G., van Boom., J.H., Altona, C., and Haasnoot, C.A.G. (1983) Nucleic Acids Research 11, 2839-2856] to explain the contrast in 13C spectroscopic properties of ApUpG in comparison to ApApG and ApApA.

Adenine Nucleotides↗
PubMed1985

13C-NMR of ribosyl A-A-A, A-A-G, and A-U-G. Synthesis and assignment.

The three RNA trinucleotides; ApApA, ApApG, and ApUpG, have been synthesized in sufficient quantity to obtain natural abundance 13C(1H)-NMR spectra at strand concentrations between 4 and 100 mM. Comparisons between 70 degrees C spectra of the three trimers and their consistuent dimers ApA, ApG, ApU, and UpG allow secure assignments to be made for most of the resonances. This paper describes the syntheses and 13C assignments of the oligomers.

Adenine Nucleotides↗
PubMed1982

On the cooperative and noncooperative binding of ethidium to DNA.

The equilibrium binding of ethidium bromide to native DNAs and to poly(dG-dC) X poly(dG-dC) has been studied by both phase partition and direct spectrophotometric techniques. The binding isotherms obtained from both experimental techniques show that ethidium binds in a cooperative manner to E. coli DNA. On the other hand, no evidence of cooperative binding was observed in the binding isotherms obtained with calf thymus, C. perfringens, M. lysodeikticus, or poly(dG-dC) X (dG-dC) under the experimental conditions used (0.1 M NaCl).

Animals↗
PubMed1981

Equilibrium binding of carcinogens and antitumor antibiotics to DNA: site selectivity, cooperativity, allosterism.

The equilibrium binding of the carcinogens N-hydroxy-N-acetyl-2-amino-fluorene (HAAF) and 4-nitroquinoline-1-oxide (NQO) to phi X174RF DNA have been studied by phase partition techniques. Both molecules bind in a cooperative manner with only a few carcinogen molecules binding to each phi X174RF DNA molecule. The binding data for both HAAF and NQO fit a model in which two carcinogens cluster into a small number of sites--four sites for HAAF and twelve sites for NQO. Phase partition techniques were also used to study the binding of actinomycin D to both calf thymus DNA and poly (dG-dC) . poly (dG-dC) at much lower r values than had been previously reported. These data exhibit humped Scatchard plots which are indicative of cooperative binding; the overall shape of the Scatchard plots are consistent with a model for drug induced allosteric transitions in the DNA structure. The cooperativity in the actinomycin D binding to calf thymus DNA increases with decreasing sodium chloride concentration, suggesting a role for DNA flexibility in allosteric binding.

2-Acetylaminofluorene↗
PubMed1979

Interactions of 4-nitroquinoline 1-oxide with deoxyribodinucleotides.

The interactions of 4-nitroquinoline 1-oxide (NQO), a potent mutagen and carcinogen, with several self- and non-self-complementary deoxydinucleotides were probed by using absorption spectra of the charge transfer bands and 1H and 13C NMR spectra. Absorption spectra were analyzed by using Benesi-Hildebrand-type equations to yield stoichiometries and equilibrium constants of complex formation. Non-self complementary dimers form weak l:1 complexes [dpTpG:NQO, K(25 degrees C) = 22 M-1] while self-complementary dimers form strong 2:1 complexes [dpCpG)2:NQO, K(25 degrees C) = 2.2 X 10(4) M-2]. A mixture of dpTpG and dpCpA with NQO gives a 2:1 complexes [dpCpG)2:NQO, K(25 degrees C) = 2.2 X 10(4) M-2]. A mixture of dpTpG and dpCpA, K(25 degrees C) = 8.6 X 10(3) M-2]. Analyses of the changes in 13C and 1H NMR chemical shifts with complex formation gave approximate orientations for the intercalation of NQO with self-complementary dimer minihelixes. In the (dpCpG)2:NQO and (dpGpC)2:NQO complexes, the NO2 group of NQO probably lies in the major grove and the NO2, NO containing NQO ring is stacked near the purine imidazole ring. In the (dpTpA)2:NQO and (dpApT)2NQO complexes, the NO2 seems to project into the minor grove and the NQO benzenoid ring is over the purine imidazole ring.

4-Nitroquinoline-1-oxide↗
PubMed1978

Interactions of 4-nitroquinoline 1-oxide with four deoxyribonucleotides.

The interactions of 4-nitroquinoline 1-oxide (NQO) with the four 5'-deoxyribonucleotides were probed using absorption spectra of the charge transfer bands and 1H and 13C nuclear magnetic resonance (NMR) spectra of nucleotide-NQO mixtures. Spectral data yielded equilibrium constants (K(dpG:NQO) = 16 M-1, K(dpA:NQO) = 12 M-1, K(dpT:NQO) = K(dpC:NQO) = 4 M-1) which suggest the preference of NQO for the guanine residue in a DNA. This is in agreement with the data of Okano, T., et al. [(1969) Gann 60, 295]. From 13C and 1H NMR data on nucleosides, a structure for the dpG:NQO complex is proposed.

4-Nitroquinoline-1-oxide↗