G2 chromosomal radiosensitivity in families with ataxia-telangiectasia

Summary Ataxia-telangiectasia (A-T) is an autosomal recessive disease involving chromosomal instability, susceptibility to cancer and X-ray hypersensitivity. The latter two features are expressed to a limited extent in the heterozygous carriers of A-T mutations. Although fibroblast lines from A-T heterozygotes clearly show increased susceptibility to the lethal effect of X-irradiation, the difference in post-irradiation survival between cell lines and normal controls is not always large enough to allow the use of X-ray sensitivity as a laboratory assay for carrier detection in A-T. Recently, we have shown in a blind study, that the extent of chromatid damage induced in the G₂ phase of the cell cycle by moderate doses of X-rays is markedly higher in A-T heterozygous cells than in normal controls. We have now applied this test to 6 additional obligatory heterozygotes and 24 individuals with different risks of being A-T carriers, from three Israeli A-T families. All 6 cell lines from the obligatory heterozygotes showed the typical hypersensitivity to the clastogenic action of X-rays in G₂; of the 24 cell lines with unknown A-T genotype, 16 showed the same hypersensitivity, and 8 responded in a normal way. The proportion of cell lines showing the A-T-heterozygous phenotype was in accord with the expected value, based on Mendelian chance calculations. Since these observations were made, a daughter of two hypersensitive parents in one of these families has been diagnosed as having A-T. This confirmed the presumed A-T heterozygosity of the parents, as indicated by the laboratory assay.     

Selective Removal of Individual Disulfide Bonds within a Potato Type II Serine Proteinase Inhibitor from Nicotiana alata Reveals Differential Stabilization of the Reactive-Site Loop

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K_i of < 5 nM, C4A/C41A-T1 has a weaker K_i of ∼ 350 nM, and the potency of the C8A/C37A variant is further decreased to a K_i of ∼ 1.8 μM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.     

Detection of heterozygous carriers of the ataxia-telangiectasia (ATM) gene by G2 phase chromosomal radiosensitivity of peripheral blood lymphocytes

In ataxia-telangiectasia (A-T) patients, mutations in a single gene, ATM, result in an autosomal recessive syndrome that embraces a variety of clinical features and manifests extreme radiosensitivity and a strong predisposition to malignancy. Heterozygotes for the ATM gene have no clinical expression of A-T but may be cancer prone with a moderate increase in in vitro radiosensitivity. We performed a blind chromosomal analysis on G₂-phase lymphocytes from 7 unrelated A-T patients, 13 obligate A-T heterozygotes (parents of the patients), and 14 normal controls following X-irradiation with 1 Gy in order to evaluate this cytogenetic method as a tool for detection of ATM carriers. Both A-T homozygotes and heterozygotes showed significantly increased levels of radiation-induced chromatid damage relative to that of normal controls. These results show that the G₂-phase chromosomal radiosensitivity assay can be used for the detection of A-T heterozygotes. In combination with molecular genetic analyses, this test may be of value in studies of familial and sporadic cancers aimed at determination of the potential involvement of ATM mutations in tumor risk or development. Received: 5 May 1997 / Accepted: 26 August 1997     

Effect of caffeine in G2 on X-ray-induced chromosomal aberrations and mitotic inhibition in ataxia telangiectasia fibroblast and lymphoblastoid cells

Summary The effect of post-treatments with caffeine in G₂ on the frequency of X-ray-induced chromatid aberrations was studied in normal and ataxia telangiectasia (A-T) fibroblast and lymphoblastoid cells. Caffeine was found to potentiate the X-ray-induced aberration yield in both normal fibroblast and lymphoblastoid cells. An enhancement was also observed in A-T lymphoblastoid cells, whereas the X-ray-induced aberration frequency in A-T fibroblasts was unaffected by the presence of caffeine. The influence of caffeine on the radiationinduced mitotic inhibition was investigated in normal and A-T fibroblasts; in both types of cell less inhibition was obtained in the presence of caffeine.     

A molecular mechanical study of netropsin-DNA interactions

We present molecular mechanical calculations on the complexes of netropsin with dA₆·dT₆, d(TATATA)₂, d(CGCGCG)₂, and d(CGCGAATTCGCG)₂. The complexes were model built using computer graphics and then completely energy refined. Our calculations are consistent with the observed AT preference for netropsin and suggest that mixed sugar pucker geometries should be more stable than uniform in netropsin complexes with poly[d(A-T)]·poly[d(A-T)] and poly(dA)·poly(dt). The netropsin·d(TATATA) and netropsin·dA₆·dT₆ complexes are significantly different in structure, leading to a possible reason why the observed thermodynamics of netropsin-association with poly[d(A-T)]·poly[d(A-T)] and with poly(dA)·poly(dT) are so different. We also model built and energy refined a structure of netropsin-d(CGCGAATTCGCG)₂ using as a guide the nmr data of Patel [(1982) Proc. Natl. Acad. Sci. USA, 79 , 6424–6428] and found a three-dimensional structure qualitatively consistent with the NOE enhancements observed by him. After our calculations were completed, we learned of an x-ray structure of a netropsin:d(CGCGAATTCGCG)₂ complex, and we compared the structure found in our calculation with the x-ray structure.     

Structural and functional heterogeneity of single-stranded DNA-binding proteins from calf thymus

A new purification technique for ‘single-stranded DNA-binding proteins’ from calf thymus permits the demonstration of a considerable heterogeneity within these proteins. Several molecular species are obtained with M_r between 24·10³ and 30·10³ and pI values between 6 and 8, showing significant differences with regard to the following functional properties: (1) strength of binding to single-stranded DNA; (2) lowering of melting temperature of poly[d(A-T)]; (3) stimulation of DNA polymerase α on a poly[d(A-T)] template. Analysis of trypsin digestion products demonstrates that the different molecular species share extensive primary sequence homology. Experiments with antibodies show that the different molecular species are antigenically related and that a 31 kDa protein present in low amounts in our preparations is very cross-reactive.     

Spectroscopic studies of 9-hydroxyellipticine binding to DNA

The binding of 9-hydroxyellipticine to calf thymus DNA, poly[d(A-T)]₂, and poly-[d(G-C)]₂ has been studied in detail by means of CD, linear dichroism, resonance light scattering, and molecular dynamics. The transition moment polarizations of 9-hydroxyelliptiycine were determined in polyvinyl alcohol stretched film. Spectroscopic solution studies of the DNA/drug complex are combined with theoretical CD calculations using the final 50 ps of a series of molecular dynamics simulations as input. The spectroscopic data shows 9-hydroxyellipticine to adopt two main binding modes, one intercalative and the other a stacked binding mode involving the formation of drug oligomers in the DNA major groove. Analysis of the intercalated binding mode in poly[d(A-T)]₂ suggests the 9-hydroxyellipticine hydroxyl group lies in the minor groove and hydrogen bonds to water with the pyridine ring protruding into the major groove. The stacked binding mode was examined using resonance light scattering and it was concluded that the drug was forming small oligomer stacks rather than extended aggregates. Reduced linear dichroism measurements suggested a binding geometry that precluded a minor groove binding mode where the plane of the drug makes a 45° angle with the plane of the bases. Thus it was concluded that the drug stacks in the major groove. No obvious differences in the mode of binding of 9-hydroxyellipticine were observed between different DNA sequences; however, the stacked binding mode appeared to be more favorable for calf thymus DNA and poly[d(G-C)]₂ than for poly[d(A-T)]₂, an observation that could be explained by the slightly greater steric hindrance of the poly[d(A-T)]₂ major groove. A strong concentration dependence was observed for the two binding modes where intercalation is favored at very low drug load, with stacking interactions becoming more prominent as the drug concentration is increased. Even at DNA : drug mixing ratios of 70:1 the stacked binding mode was still important for GC-rich DNAs. © 1998 John Wiley & Sons, Inc. Biopoly 46: 127–143, 1998     

Clearly differentiated and stable chromosome bands produced by a spermine bis-acridine, a bifunctional intercalating analogue of quinacrine

Characteristic fluorescent banding patterns on human metaphase chromosomes are produced by treating chromosome preparations directly with a spermine bis-acridine fluorochrome (CMA)₂S. The clearly differentiated bands are similar to those produced by quinacrine (Q-banding), but show enhanced definition between bright and dull regions as compared with the banding patterns obtained by the quinacrine technique. In addition, the bands on chromosomes produced by (CMA)₂S show insignificant fluorescence fading over extended periods of excitation. Solution interactions between DNA and (CMA)₂S showed a greater fluorescence differential between fluorescence enhancement by the alternating polymers poly d(A-T) · poly d(A-T) and fluorescence quenching by the polynucleotide poly d(G-C) · poly d(G-C) for this fluorochrome than was observed for quinacrine. The increased definition in Q-type bands produced by the spermine bis-intercalating derivative and the lack of fluorescence fading make this fluorochrome an excellent one for routine clinical cytogenetic analysis.     

Physical association of two simple alkylators to some DNA sequences

Molecular mechanics calculations have been used to determine the preferred physical association sites of the known alkylating agent dimethyl aziridinium ion (Az⁺) and a CH prototype test probe with B-form, tetrameric DNA sequences. Electrostatic interactions are most important in determining these preferential physical association sites. In turn, the intermolecular energy minima depend on the charge distribution assigned to the DNA sequence. However, for three reported DNA charge distributions, only two distinct sets of energy minima were obtained for the CH-like ion interacting with (G-C)₄, (A-T)₄, and [(G-C)·(A-T)]₂ deoxyribonucleic acids. These minima correspond to physical association geometries in which the CH-like ion is near known alkylation sites. The results of the Az⁺ … [(G-C)·(A-T)]₂ interaction are virtually identical to those found for the CH-like ion. Aqueous solvation energetics have little effect on the physical association of Az⁺ with [(G-C)·(A-T)]₂.     

Conformational properties of poly[d(G-T)].poly[d(C-A)] and poly[d(A-T)] in low- and high-salt solutions: NMR and laser Raman analysis

³¹P- and ¹H-nmr and laser Raman spectra have been obtained for poly[d(G-T)]·[d(C-A)] and poly[d(A-T)] as a function of both temperature and salt. The ³¹P spectrum of poly[d(G-T)]·[d(C-A)] appears as a quadruplet whose resonances undergo separation upon addition of CsCl to 5.5M. ¹H-nmr measurements are assigned and reported as a function of temperature and CsCl concentration. One dimensional nuclear Overhauser effect (NOE) difference spectra are also reported for poly[d(G-T)]·[d(C-A)] at low salt. NOE enhancements between the H8 protons of the purines and the C5 protons of the pyrimidines, (H and CH₃) and between the base and H-2′,2″ protons indicate a right-handed B-DNA conformation for this polymer. The NOE patterns for the TH3 and GH1 protons in H₂O indicate a Watson–Crick hydrogen-bonding scheme. At high CsCl concentrations there are upfield shifts for selected sugar protons and the AH2 proton. In addition, laser Raman spectra for poly[d(A-T)] and poly[d(G-T)]·[d(C-A)] indicate B-type conformations in low and high CsCl, with predominantly C2′-endo sugar conformations for both polymers. Also, changes in base-ring vibrations indicate that Cs⁺ binds to O2 of thymine and possibly N3 of adenine in poly[d(G-T)]·[d(C-A)] but not in poly[d(A-T)]. Further, ¹H measurements are reported for poly[d(A-T)] as a function of temperature in high CsCl concentrations. On going to high CsCl there are selective upfield shifts, with the most dramatic being observed for TH1′. At high temperature some of the protons undergo severe changes in linewidths. Those protons that undergo the largest upfield shifts also undergo the most dramatic changes in linewidths. In particular TH1′, TCH₃, AH1′, AH2, and TH6 all undergo large changes in linewidths, whereas AH8 and all the H-2′,2″ protons remain essentially constant. The maximum linewidth occurs at the same temperature for all protons (65°C). This transition does not occur for d(G-T)·d(C-A) at 65°C or at any other temperature studied. These changes are cooperative in nature and can be rationalized as a temperature-induced equilibrium between bound and unbound Cs⁺, with duplex and single-stranded DNA. NOE measurements for poly[d(A-T)] indicate that at high Cs⁺ the polymer is in a right-handed B-conformation. Assignments and NOE effects for the low-salt ¹H spectra of poly[d(A-T)] agree with those of Assa-Munt and Kearns [(1984) Biochemistry 23 , 791–796] and provide a basis for analysis of the high Cs⁺ spectra. These results indicate that both polymers adopt a B-type conformation in both low and high salt. However, a significant variation is the ability of the phosphate backbone to adopt a repeat dependent upon the base sequence. This feature is common to poly[d(G-T)]·[d(C-A)], poly[d(A-T)], and some other pyr–pur polymers [J. S. Cohen, J. B. Wouten & C. L Chatterjee (1981) Biochemistry 20 , 3049–3055] but not poly[d(G-C)].     

Molecular heterogeneity of platelet-activating factor produced by stimulated human polymorphonuclear leukocytes

The molecular heterogeneity of platelet-activating factor (PAF) produced by stimulated human neutrophilic polymorphonuclear leukocytes (PMN) was assessed by both normal and reverse phase high performance liquid chromatography (HPLC). As detected by rabbit platelet stimulation, at least 5 PAF molecules were separated by HPLC. Fast atom bombardment (FAB) mass spectrometry revealed one of these PAFs was acetyl glyceryl ether phosphorylcholine (AGEPC) with a C_16:0 alkyl chain in the $\text{sn}$-1 position. Although the structures of the remaining PAFs are unknown, two of the peaks of PAF activity had the same retention times on reverse phase HPLC as the C₁₅- and C₁₈-saturated alkyl chain AGEPC homologues. These studies indicate that the human PMN produces multiple molecular species of PAF.     

Alkaline opening of imidazole ring of 7-methylguanosine. 1. Analysis of the resulting pyrimidine derivatives

Column chromatography and spectroscopy have been employed in analyzing pyrimidine derivatives obtained from alkaline-treated 7-methylguanosine (7-meGuo). High performance liquid chromatography (HPLC) revealed that the alkaline generated products consist predominantly of two forms of ring opened 7-methylguanine (rom⁷Gua) in equal amounts. Material from both Dowex 50 and Sephadex LH-20 columns was readily resolvable into two HPLC peaks. The species in one peak appears to be composed of formylated and that in the other of deformylated rom⁷Gua. The presence of a deformylated species is supported by the absence of radioactivity in one of the two peaks obtained when ring opened [8-¹⁴C]guanosine was analyzed by HPLC. The formylated species was retained on the liquid chromatography column for 8 min with a 3% methanol, 0.01 M NH₄H₂PO₄ (pH 5.1) solvent and for 6 min with a 6% methanol, 0.01 N NH₄H₂PO₄ (pH 5.1) solvent system; the deformylated species was retained for 6.3 min with the first solvent and 4.5 min with the second solvent. Subsequent to Dowex 50 chromatography in an ammonium formate solvent, about 90% of the material was formylated. When stored at 24°C for 72 h in a solvent without formate ions, the material was shown by HPLC to consist of equal amounts of the formylated and deformylated species. These results indicate that the two species of rom⁷Gua are in equilibrium. The rom⁷Gua excised from DNA by formamidopyrimidine (FAPy)-DNA glycosylase was shown to coelute with the formylated species.     

Reversed-phase liquid chromatographic analysis of hydrophobic interaction between proanthocyanidins and a C8-alkyl compound in aqueous solution

Structural and physicochemical properties of oligomeric flavan-3-ols (proanthocyanidins) in aqueous solution were investigated by spectrometric and reversed-phase (RP) HPLC analyses. Circular dichroism and fluorescence spectra of (–)-epicatechin (EC) oligomers linked through C-4 to C-8 interflavan bonds showed that EC oligomers larger than dimers formed a stable secondary structure in water. These EC oligomers are water-soluble hydrophilic compounds, whereas the oligomers were strongly retained by a C₈-alkyl stationary phase under conventional RP-HPLC conditions. In a further C₈-HPLC study, the hydrophobic interaction between EC oligomers and 1-octanesulfonic acid sodium salt (OSA Na) added to the mobile phase was quantitatively evaluated based on the relationship between the logarithm of the retention factor of the solute and the OSA Na concentration in the mobile phase. The strength values of the hydrophobic interaction of EC oligomers larger than dimers were the highest of 22 tested polyphenolic standards.     

Influence of acetylcysteine on cytogenetic effects of etoposide in mouse oocytes

The influence of N-acetylcysteine (ACC) on the cytogenetic effects of etoposide in F₁ CBA × C57BL/6 mice was studied. Etoposide introduced intraperitoneally in doses of 10, 20, 40, and 60 mg/kg has a dose-dependent clastogenic activity and has an aneugenic effect with the induction of mainly hypohaploid oocytes. ACC significantly decreases the aneugenic and clastogenic activity of etoposide (20 mg/kg) in oocytes of 6-, 9-, and 12-week-old mice during triple introduction at a dose 200 mg/kg per os. The most pronounced anticlastogenic ACC activity (an 80% decrease) was registered in 9-week-old females; a 100% decrease in aneugenesis was detected in 6-week-old female mice.     

Familial Mediterranian fever: clastogenic plasma factors correlated with increased O2 –– production by neutrophils

Familial Mediterranean fever (FMF) is an autosomal recessive disease predominantly affecting Armenians and non-Ashkenazi Jews. The disease begins in childhood with paroxysmal attacks of pain and fever accompanied by peritonitis, pleuritis, and synovitis. During the acute phase, there is a massive influx of polymorphonuclear leukocytes into the serosal membranes, connected with degranulation of the neutrophils and with secretion of lysosomal enzymes and pyrogenic substances. An increase in the lipoxygenase product, leukotriene B₄, a chemotactic agent, and a decrease in the activity of the inhibitor of chemotaxis, C5a, in serosal fluids have been considered responsible. Previous work from our laboratories had shown that the chromosomal instability observed in blood cultures of patients with FMF is secondary to circulating clastogenic factors (CFs), and that the antioxidant enzyme superoxide dismutase, as well as lipoxygenase inhibitors, reduce the chromosome damaging effects. CFs are observed in chronic inflammatory diseases and in various other pathological conditions accompanied by oxidative stress. Similar clastogenic materials were found in supernatants of neutrophils and monocytes after a respiratory burst and were shown to contain lipid peroxidation products and cytokines. In the present study we compared the clastogenic effects exerted by plasma ultrafiltrates from 20 adult patients with FMF to the unstimulated O₂ ^– production of their neutrophils. In comparison to 20 age- and sex-matched controls, which were studied simultaneously, the O₂ ^– production by patient’s neutrophils was routinely higher than that of controls. The clastogenic effects of patient’s plasma, expressed as the number of chromosomal aberrations induced in test cultures of healthy donors, were correlated with the importance of O₂ ^– production by their neutrophils (r = 0.5235). Even if the relative contribution of disturbance in arachidonic acid metabolism, neutrophil activation, and CF formation in the disease process remains unclear, the demonstration of oxidative stress in this genetic disorder suggests the use of antioxidants and free radical scavengers, in particular during acute attacks, when the classical colchicine treatment is without effect. Received: 15 June 1997 / Accepted: 18 July 1997     

Novel Hoogsteen-like Bases for Recognition of the C-G Base Pair by DNA Triplex Formation

Effective sequence-specific recognition of duplex DNA is possible by triplex formation with natural oligonucleotides via Hoogsteen H-bonding. However, triplex formation is in practice limited to pyrimidine oligonucleotides that bind duplex A-T or G-C base pair DNA sequences specifically at homopurine sites in the major groove as T·A-T and C⁺ ·G-C triplets. Here we report the successful modelling of novel unnatural nucleosides that recognize the C-G DNA base pair by Hoogsteen-like major groove interaction. These novel Hoogsteen nucleotides are examined within model A-type and B-type conformation triplex structures since the DNA triplex can be considered to incorporate A-type and/or B-type configurational properties. Using the same deoxyribose-phosphodiester and base-deoxyribose dihedral angle configuration, a triplet comprised of a C-G base pair and the novel Hoogsteen nucleotide, Y2, replaces the central T·A-T triplet in the triplex. The presence of any structural or energetic perturbations due to the central triplet in the energy-minimized triplex is assessed with respect to the unmodified energy minimized (T·A-T)₁₁ starting structures. Incorporation of this novel triplet into both A-type and B-type natural triplex structures provokes minimal change in the configuration of the central and adjacent triplets.     

Additive coclastogenicity of sodium selenite and caffeine in CHO cells treated withN-methyl-N′-Nitro-N-Nitrosoguanidine

The clastogenic effect ofN-methyl-N′-nitro-N-nitrosoguanidine (MNNG) in Chinese hamster ovary (CHO) cells and its modulation by Na₂SeO₃ and caffeine were studied by metaphase analysis of chromosome aberrations (CA) as well as by measuring the formation and repair of single-strand (ss) DNA breaks employing hydroxylapatite chromatography. Treatment of CHO cells with MNNG (1.25 or 2.5 × 10-5M) for 3 h caused CA in 11 and 19% of metaphases scored, respectively. Pretreatment of cells with Na₂SeO₃ (1–5 μg/mL) or caffeine (0.2–2.0 mg/mL) for 2 h resulted in a 2–3.5-fold increase of CA frequency. Addition of both modulators during the mutagen exposure tended to cause a slight inhibition of clastogenic activity of MNNG (1.25 × 10⁻⁵ M) or had no effect on CA number when MNNG was used at a concentration of 2.5 × 10−5M. Posttreatment of CHO cells with Na₂SeO₃ for 20 h after MNNG was ineffective in influencing the number of metaphases with CA, whereas, at these conditions, caffeine enhanced up to 6-7-fold the clastogenic activity of MNNG. Addition of both modulators during the whole experiment, 2 h pretreatment included, resulted in a further significant increase of CA frequency up to the total pulverization of chromosomes in all metaphases scored. The coclastogenic effect of caffeine was greater in this case. The enhancement of chromosome-damaging activity of MNNG by selenite and caffeine was better expressed when this carcinogen was applied at the higher concentration used. An additive coclastogenic effect was observed in CHO cells treated simultaneously with Na₂SeO₃ and caffeine plus MNNG. In addition, the treatment of CHO cells with MNNG (5 × 10⁻⁶ M) caused a rapid increase of ssDNA breaks number reaching maximal values after 30–45 min. However, up to 50–60% of MNNG-induced ssDNA breaks were repaired during the first 60–150 min after the mutagen exposure. The 2 h pretreatment of CHO cells with Na₂SeO₃ (2 μg/mL) or the addition of this trace element after MNNG had no effect on formation and repair of MNNG-induced ssDNA breaks. The coclastogenic effect of Na₂SeO₃ in CHO cells treated with MNNG was not directly linked to the induction and disappearance of ssDNA breaks measured by hydroxylapatite chromatography.     

Helix coil transitions of d (A)n·d(T)n,d(A-T)n·d(A-T)n,and d(A-A-T)n·d(A-T-T)n; evaluation of parameters governing DNA stability

The thermally induced helix-coil transitions of three A-T DNAs, d(A)_n·d(T)_n, d(A-T)_n·d(A-T)_n, and d(A-A-T)_n·d(A-T-T)_n, were studied. Experimental transition curves of the DNAs were analyzed using the loop entropy model of DNA melting. The calculation of the melting curve of d(A-A-T)_n·d(A-T-T)_n is presented using the integral equation formalism of Goel and Montroll. The aim of this work was to evaluate thermodynamic parameters which govern DNA stability and to test the theoretical model employed in the analysis. Our results show (1) an excellent over-all agreement between theory and experiment, (2) a loop entropy exponent k = 1.55 ± 0.05 provided the best fit to all the polymer transition curves, (3) the evaluated stacking free energies reflect the relative stability of the DNAs, and (4) the stacking energies of the ApA·TpT dimer evaluated from d(A)_n·d(T)_n and d(A-A-T)_n·d(A-T-T)_n differ. The last result is consistent with different conformations for the dimer in these two polymers.     

Dissymmetric recognition of the helical sense of deoxyribonucleic acid and evidence for binding of reporter molecules from the minor groove of DNA

Through the utilization of optically active DNP-derivatives of l- and d-proline, evidence is presented which suggests that nucleic acids exist as right-handed helices in solution. The results of ultraviolet absorption, circular dichroism, proton magnetic resonance (pmr), T_m of the helix-coil transition, viscometric, and binding studies are consistent with the above interpretation. It is shown that several types of DNA (i.e., salmon sperm, calf thymus, Micrococcus luteus, poly d(A-T)-poly d(A-T) and poly d(I-C)-poly d(I-C)) exist in a right-handed helical structure in solution. In addition, evidence is presented which strongly indicates that the 2,4-dinitroaniline ring of DNP-proline is intercalated between base-pairs of DNA and the prolyl side chain situated in the minor groove. Moreover, it is shown that the more sterically hindered DNP-derivatives exhibit a higher selectivity for A-T binding sites.