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E Tuite

Publications and source records attributed to E Tuite.

13 recordsLinked to original sources
PubMed2001

Photobleaching of asymmetric cyanines used for fluorescence imaging of single DNA molecules.

The photobleaching of the cyanine dyes YO and YOYO has been investigated for both free and DNA-bound dyes, using absorption and fluorescence spectroscopy coupled with fluorescence microscopy. For the free dyes, the nature of the reactive species involved in the photodegradation process is different for the monomer and the dimer, as shown by scavenger studies. For DNA-bound dyes, photoinduced fading of the visible absorption band occurs by different pathways depending on the drug binding mode and can be attenuated by appropriate scavengers. However, none of these scavengers were found to have any significant effect on the photobleaching of dye fluorescence. It appears that the reduction of fluorescence intensity comes from a quenching of the dye fluorescence by modified DNA bases, possibly 8-oxo-7,8-dihydro-2'-deoxyguanosine.

Animals↗
PubMed2000

Thermodynamics of sequence-specific binding of PNA to DNA.

For further characterization of the hybridization properties of peptide nucleic acids (PNAs), the thermodynamics of hybridization of mixed sequence PNA-DNA duplexes have been studied. We have characterized the binding of PNA to DNA in terms of binding affinity (perfectly matched duplexes) and sequence specificity of binding (singly mismatched duplexes) using mainly absorption hypochromicity melting curves and isothermal titration calorimetry. For perfectly sequence-matched duplexes of varying lengths (6-20 bp), the average free energy of binding (DeltaG degrees ) was determined to be -6.5+/-0.3 kJ mol(-1) bp(-1), corresponding to a microscopic binding constant of about 14 M(-1) bp(-1). A variety of single mismatches were introduced in 9- and 12-mer PNA-DNA duplexes. Melting temperatures (T(m)) of 9- and 12-mer PNA-DNA duplexes with a single mismatch dropped typically 15-20 degrees C relative to that of the perfectly matched sequence with a corresponding free energy penalty of about 15 kJ mol(-1) bp(-1). The average cost of a single mismatch is therefore estimated to be on the order of or larger than the gain of two matched base pairs, resulting in an apparent binding constant of only 0.02 M(-1) per mismatch. The impact of a mismatch was found to be dependent on the neighboring base pairs. To a first approximation, increasing the stability of the surrounding region, i.e., the distribution of A.T and G.C base pairs, decreases the effect of the introduced mismatch.

Base Pair Mismatch↗
PubMed1999

Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA--DNA complex by inactive nucleotides.

The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA-DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA-DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5'-O-3-thiotriphosphate or guanosine 5'-O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA-DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex.

Circular Dichroism↗
PubMed1998

Hybridization of peptide nucleic acid.

The thermodynamics of hybridization and the conformations of decameric mixed purine-pyrimidine sequence PNA/PNA, PNA/DNA, and DNA/DNA duplexes have been studied using fluorescence energy transfer (FET), absorption hypochromicity (ABS), isothermal titration calorimetry (ITC), and circular dichroism (CD) techniques. The interchromophoric distances determined in the FET experiments on fluorescein- and rhodamine-labeled duplexes indicate that the solution structures of the duplexes are extended helices in agreement with available NMR (PNA/DNA) and crystal X-ray data (PNA/PNA). The melting thermodynamics of the duplexes was studied with both FET and ABS. The thermodynamic parameters obtained with ABS are in good agreement with the parameters from calorimetric measurements while FET detection of duplex melting gives in most cases more favorable free energies of hybridization. This discrepancy between FET and ABS detection is ascribed to the conjugated dyes which affect the stability of the duplexes substantially. Especially, the dianionic fluorescein attached via a flexible linker either to PNA or to DNA seems to be involved in an attractive interaction with the opposite dicationic lysine when hybridized to a PNA strand. This interaction leads to an increased thermal stability as manifested as a 3-4 degreesC increase of the melting temperature. For the PNA/DNA duplex where fluorescein is attached to the PNA strand, a large destabilization (DeltaTm = -12 degreesC) occurs relative to the unlabeled duplex, probably originating from electrostatic repulsion between the fluorescein and the negatively charged DNA backbone. In the case of the PNA/PNA duplex, the sense of helicity of the duplex is reversed upon conjugation of fluorescein via a flexible linker arm, but not when the fluorescein is attached without a linker to the PNA.

Circular Dichroism↗
PubMed1997

Nucleotide cofactor-dependent structural change of Xenopus laevis Rad51 protein filament detected by small-angle neutron scattering measurements in solution.

Rad51 protein, a eukaryotic homologue of RecA protein, forms a filamentous complex with DNA and catalyzes homologous recombination. We have analyzed the structure of Xenopus Rad51 protein (XRad51.1) in solution by small-angle neutron scattering (SANS). The measurements showed that XRad51.1 forms a helical filament independently of DNA. The sizes of the cross-sectional and helical pitch of the filament could be determined, respectively, from a Guinier plot and the position of the subsidiary maximum of SANS data. We observed that the helical structure is modified by nucleotide binding as in the case of RecA. Upon ATP binding under high-salt conditions (600 mM NaCl), the helical pitch of XRad51.1 filament was increased from 8 to 10 nm and the cross-sectional diameter decreased from 7 to 6 nm. The pitch sizes of XRad51.1 are similar to, though slightly larger than, those of RecA filament under corresponding conditions. A similar helical pitch size was observed by electron microscopy for budding yeast Rad51 [Ogawa, T., et al. (1993) Science 259, 1896-1899]. In contrast to the RecA filament, the structure of XRad51.1 filament with ADP is not significantly different from that with ATP. Thus, the hydrolysis of ATP to ADP does not modify the helical filament of XRad51.1. Together with our recent observation that ADP does not weaken the XRad51.1/DNA interaction, the effect of ATP hydrolysis on XRad51.1 nucleofilament should be very different from that on RecA.

Adenine Nucleotides↗
PubMed1997

Effects of minor and major groove-binding drugs and intercalators on the DNA association of minor groove-binding proteins RecA and deoxyribonuclease I detected by flow linear dichroism.

Linear and circular dichroic spectroscopies have been employed to investigate the effects of small DNA ligands on the interactions of two proteins which bind to the minor groove of DNA, viz. RecA protein from Escherichia coli and deoxyribonuclease I (bovine pancreas). Ligands representing three specific non-covalent binding modes were investigated: 4',6-diamidino-2-phenylindole and distamycin A (minor groove binders), methyl green (major groove binder), and methylene blue, ethidium bromide and ethidium dimer (intercalators). Linear dichroism was demonstrated to be an excellent detector, in real time, of DNA double-strand cleavage by deoxyribonuclease I. Ligands bound in all three modes interfered with the deoxyribonuclease I digestion of dsDNA, although the level of interference varied in a manner which could be related to the ligand binding site, the ligand charge appearing to be less important. In particular, the retardation of deoxyribonuclease I cleavage by the major groove binder methyl green demonstrates that accessibility to the minor groove can be affected by occupancy of the opposite groove. Binding of all three types of ligand also had marked effects on the interaction of RecA with dsDNA in the presence of non-hydrolyzable cofactor adenosine 5'-O-3-thiotriphosphate, decreasing the association rate to varying extents but with the strongest effects from ligands having some minor groove occupancy. Finally, each ligand was displaced from its DNA binding site upon completion of RecA association, again demonstrating that modification of either groove can affect the properties and behaviour of the other. The conclusions are discussed against the background of previous work on the use of small DNA ligands to probe DNA-protein interactions.

Circular Dichroism↗
PubMed1997

Binding mode of [ruthenium(II) (1,10-phenanthroline)2L]2+ with poly (dT*dA-dT) triplex. Ligand size effect on third-strand stabilization.

The binding of homochiral [Ru(II)(1,10-phenanthroline)2L]2+ complexes [where [L = 1,10-phenanthroline (phen), dipyrido[3,2-a:2',3'-c]phenazine (DPPZ) or benzodipyrido[3,2-a:2',3'-c]phenazine (BDPPZ)] to poly(dT*dA-dT) triplex has been investigated by linear and circular dichroism and thermal denaturation. Analysis of the linear dichroism spectra indicates that the extended DPPZ and BDPPZ ligands lie approximately parallel to the base-pair and base-triplet planes consistent with intercalation which is also supported by strong hypochromism in the interligand absorption bands with either duplex or triplex. The spectral properties of any of the metal complex enantiomers were similar for binding to either duplex or triplex DNA, indicating that the third strand, which occupies the major groove of the template duplex, has little effect on the binding geometries and hence supports the hypothesis that the metal complexes all bind from the minor groove with the DPPZ and BDPPZ ligands intercalated but without intercalation in the case of [Ru(phen)3]2+. Third-strand stabilization depended on the nature of the third substituted phenanthroline chelate ligand but was not directly related to its size, with stabilizing power increasing in the order phen < BDPPZ < DPPZ. This observation further supports intercalation of the extended ligands from the minor groove of the triplex since the extended BDPPZ ligand that would protrude into the major groove of the template would have greater steric interference than DPPZ with the third DNA strand.

Circular Dichroism↗
PubMed1996

Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO.

Photocleavage of dsDNA by the fluorescent DNA stains oxazole yellow (YO), its dimer YOYO) and the dimer TOTO of thiazole orange (TO) has been investigated as a function of binding ratio. On visible illumination, both YO and YOYO cause single-strand cleavage, with an efficiency that varies with the dye/DNA binding ratio in a manner which can be rationalized in terms of free dye being an inefficient photocleavage reagent and externally bound dye being more efficient than intercalated dye. Moreover, the photocleavage mechanism changes with binding mode. Photocleavage by externally bound dye is, at least partly, oxygen dependent with scavenger studies implicating singlet oxygen as the activated oxygen intermediate. Photocleavage by intercalated dye is essentially oxygen-independent but can be inhibited by moderate concentrations of beta- mercaptoethanol--direct attack on the phosphoribose backbone is a possible mechanism. TOTO causes single-strand cleavage approximately five times less efficiently than YOYO. No direct double-strand breaks (dsb) are detected with YO or YOYO, but in both cases single-strand breaks (ssb) are observed to accumulate to eventually produce double-strand cleavage. With intercalated YO the accumulation occurs in a manner consistent with random generation of strand lesions, while with bisintercalated YOYO the yield of double-strand cleavage (per ssb) is 5-fold higher. A contributing factor is the slow dissociation of the bis-intercalated dimer, which allows for repeated strand-attack at the same binding site, but the observation that the dsb/ssb yield is considerably lower for externally bound than for bis-intercalated YOYO at low dye/DNA ratios indicates that the binding geometry and/or the cleavage mechanism are also important for the high dsb-efficiency. In fact, double-strand cleavage yields with bis-intercalated YOYO are higher than those predicted by simple models, implying a greater than statistical probability for a second cleavage event to occur adjacent to the first (i.e. to be induced by the same YOYO molecule). With TOTO the efficiency of the ssb-accumulation is comparable to that observed with YOYO.

Bacteriophage phi X 174↗
PubMed1995

Effects of intercalators on complexation of RecA with duplex DNA.

To elucidate the binding mode of recombination protein A (RecA) to double-stranded (ds) DNA, the effects on the RecA-DNA interaction of several mono- and bisintercalators of the acridine, phenanthridine, and cyanine classes have been investigated by linear dichroism spectroscopy. Simple monointercalators lacking side chains efficiently promoted the binding of RecA to dsDNA in the absence of nucleotide cofactor, which is otherwise required. Bisintercalators varied in their ability to induce RecA binding, while monointercalators with aminoalkyl side chains proved inefficient. Modification of DNA structure by the intercalator appears to be necessary for induction of RecA binding, but if the intercalator has a bulky minor-groove-binding side chain, it does not induce RecA binding. In detailed studies with acridines, neither the binding geometry of intercalators nor the structure of DNA was significantly modified upon binding of RecA without cofactor. Judged by circular dichroism, similar RecA conformational changes accompanied bis-9-aminoacridine- and ATP gamma S-induced RecA association with DNA. In the presence of ATP gamma S, the intercalators inhibited the rate of RecA binding to dsDNA and were extruded from DNA upon binding of RecA. This competitive aspect may suggest that intercalation of some amino acid residue(s) plays a role in nucleotide-induced RecA binding. The stoichiometry of the RecA-DNA-intercalator filament was determined; in the fully formed filament the base pair:intercalator ratio is 2, and the base pair:RecA ratio also 2. This contrasts with a base pair:RecA ratio of 3 in the ATP gamma S-induced filament, although in both cases the DNA experiences 50% extension.

Acridines↗
PubMed1995

Intercalative interactions of ethidium dyes with triplex structures.

The binding of phenanthridine dyes to triplex poly(dT)*poly(dA).poly(dT) and its precursor duplex poly(dA).poly(dT) is characterized using linear dichroism and circular dichroism spectroscopy, and thermal denaturation. The two monomeric dyes ethidium bromide and propidium iodide are shown to behave similarly to each other in intercalating into and stabilizing both the duplex and the triplex structures. However, contrary to expectations, the extra cationic side-chain of propidium iodide provides no significant extra stabilization of triplex compared with ethidium bromide, although propidium does stabilize the duplex more than ethidium. The monomeric dyes appear to have somewhat different binding geometries with the duplex and triplex polymers. The dimeric dye ethidium homodimer is found to bis-intercalate in the triplex as well as the duplex but, in contrast to the monomers, no variation in geometry between duplex and triplex is observed. However, although dimer stabilizes the duplex, it has no effect on the thermal stability of the triplex. This lack of binding preferentiality of the dimer for triplex compared with the monomeric dyes indicates greater constraints on the accommodation of a bis-intercalator in the triplex structure than in the duplex.

Circular Dichroism↗
PubMed1978

Equipment selection for inhouse laundry demands careful planning.

Laundry equipment has become increasingly sophisticated in recent years, and, as a result, equipment selection decisions have become extremely complex. Whether installing an in-house laundry or modernizing an existing one, equipment selection demands knowledgeable consideration of several key factors.

Equipment and Supplies, Hospital↗