Structure of Poly (dT)·Poly (dA)·Poly (dT)

Abstract

The molecular structure of poly (dT)·poly (dA)·poly (dT) has been determined and refined using the continuous x-ray intensity data on layer lines in the diffraction pattern obtained from an oriented fiber of the DNA. The final R-value for the preferred structure is 0.29 significantly lower than that for plausible alternatives. The molecule forms a 12-fold right- handed triple-helix of pitch 38.4 Å and each base triplet is stabilized by a set of four Crick-Watson-Hoogsteen hydrogen bonds. The deoxyribose rings in all the three strands have C2′-endo conformations. The grooveless cylindrical shape of the triple-helix is consistent with the lack of lateral organization in the fiber.     

Structures for the polynucleotide complexes poly(dA) · poly(dT) and poly(dT) · poly(dA) · poly(dT)

X-ray diffraction analyses of fibers of polydeoxyadenylic acid · polydeoxythymidylic acid show that this molecule exists as a 10-fold double-helix with axial rise per nucleotide $\text{h = 3.24}\text{to}\text{3.29}\text{A}\text{?}$. The structure is very similar to B-DNA ($\text{h = 3.37}\text{A}\text{?}$) in having C3-exo furanose rings and base-pairs positioned centrally on the helix axis, but distinctive enough to have two packing modes, neither of which has been observed for B-DNA. Although the triple-stranded poly(dT) · poly(dA) · poly(dT) also has a large value of h(3.26 Å), each of the chains is a 12-fold helix of the A-genus with C3-endo furanose rings and bases displaced several Angstrom units from the helix axis.     

Studies on synthetic chromatins containing poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC)

Core histones, (H2A,H2B,H3,H4)₂, were reconstituted with the synthethic polynucleotides poly(dA-dT)·poly(dA-dT) and poly(dG-dC)·poly(dG-dC) to yield synthetic chromatins containing 200 basepairs per octamer. These synthetic chromatins displayed a 36% decrease in the circular dichroism (CD) peak ellipticity from the value of the polynucleotide free in solution; the poly(dA-dT)·poly(dA-dT)/chromatin showed an increase in the complexity of the thermal denaturation profile compared to that of the polynucleotide. Both the temperature of maximum $\text{d}\text{h}\text{d}\text{T}$ for each transition (T_m) and the relative amount of poly(dA-dT)·poly(dA-dT) in the synthetic chromatin melting in each of the four thermal transitions is a function of the ionic strength over the 0–5 mM sodium phosphate range (0.25 mM EDTA, pH 7.0); a shift of material toward higher melting transitions was observed with increasing ionic strength. The CD peak ellipticity value for both synthetic chromatins was ionic strength-independent over the 0–5 mM sodium phosphate range. These results are in contrast to those observed with $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin (Fulmer, A. and Fasman, G.D. (1979) Biopolymers 18, 2875–2891), where an ionic strength dependence was found. Differences in the CD spectra between poly(dA-dT)·poly(dA-dT)/chromatin, poly(dG-dC)·poly(dG-dC)/chromatin and $\text{H}\text{1}\text{H}\text{5}$ stripped chicken erythrocyte chromatin suggest subtle differences in assembly. Finally, the temperature dependence of the CD spectra of poly(dA-dT)·poly(dA-dT)-containing synthetic chromatin, which is similar to that for the polynucleotide, suggests the core histone bound polynucleotide has a large degree of conformational flexibility allowing it to undergo the premelt transition.     

The Structure of Triple Helical Poly(U)·Poly(A)·Poly(U) Studied by Raman Spectroscopy

Abstract

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U)·poly(A) ·poly(U) triple helix. We compared the Raman spectra of poly(U)·poly(A)·poly(U) in H₂O and D₂O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A)·poly(U). The presence of a Raman band at 863 cm^?1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3′ endo; that of the second poly(U) chain may be C2′ endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A)·poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

Differential expression of ·1(IV), ·2(IV), ·5(IV) and ·6(IV) collagen chains in the basement membrane of basal cell carcinoma

Type IV collagen, the major component of basement membrane, consists primarily of ·1(IV) and ·2(IV) chains. Recently, other types of collagen IV chains, i.e. ·3(IV), ·4(IV), ·5(IV) and ·6(IV) chains, have been identified by protein chemistry and molecular cloning. We have examined the diversity of the assembly of ·(IV) chains of the basement membrane surrounding tumour nests of basal cell carcinomas, in tissues from 11 patients, by immunohistochemical analysis using specific monoclonal antibodies to six ·(IV) chain. The immunostaining profile of each chain differed with respect to the histological subtypes of basal cell carcinoma. In the morphea-like subtype, which was more invasive, ·1(IV) and ·2(IV) chains were discontinuously stained, and ·5(IV) and ·6(IV) chains were entirely absent. However, in the superficial subtype, which was non-aggressive, ·1(IV), ·2(IV), ·5(IV) and ·6(IV) chains were well stained compared with the other subtypes of basal cell carcinoma. In addition, in the solid subtype, which showed slow growth and ulceration, ·1(IV) and ·2(IV) chains were continuously stained, and ·5(IV) and ·6(IV) chains were discontinuous or absent. The assembly of ·5(IV) and ·6(IV) chains into the basement membrane was inhibited in the solid and morphea subtypes of BCC. This differential expression of type IV collagen chains seems to be associated with the invasive potential of basal cell carcinoma This revised version was published online in November 2006 with corrections to the Cover Date.     

Parallel-Stranded Oligonucleotides with Alternating d(A-isoG)/d(T·C) and d(A·G)/d(T·m5isoC) Sequences

Abstract

Parallel-stranded (ps) DNA hairpins with alternating d(A-isoG)/d(T·C) (designated as ps-t1) and d(A·G)/d(T·m⁵isoC) (ps-t2) sequences were studied by means of UV, CD and fluorescence spectroscopy. The thermostability of d(A·G)/d(T·m⁵isoC) sequence was close to that of aps d(G·A)/d(T·C). The stability of the ps d(A·isoG)/d(T·C) sequence was even higher than that of a related anti-parallel-stranded (aps) d(G·A)/d(T·C) sequence, being unique for ps DNAs studied so far.     

Interaction between double stranded poly(dA-dT)·poly (dA-dT) and lucanthone

270 MHz ¹H NMR and theoretical studies indicate that the drug lucanthone forms intercalated complexes with the synthetic DNA poly(dA-dT)·poly(dA-dT). In the intercalated complex the long axis of the drug is perpendicular to the helix axis and parallel to the base pair axis, i.e., the long axis is perpendicular to the dyad axis.     

Interaction of 9-O-(ω-amino) alkyl ether berberine analogs with poly(dT)·poly(dA)*poly(dT) triplex and poly(dA)·poly(dT) duplex: a comparative study

Isoquinoline alkaloids and their analogs represent an important class of molecules for their broad range of clinical and pharmacological utility. These compounds are of current interest owing to their low toxicity and excellent chemo preventive properties. These alkaloids can play important role in stabilising the nucleic acid triple helices. The present study has focused on the interaction of five 9-O-(ω-amino) alkyl ether berberine analogs with the DNA triplex poly(dT)·poly(dA)*poly(dT) and the parent duplex poly(dA)·poly(dT) studied using various biophysical techniques. Scatchard analysis of the spectral data indicated that the analogs bind both to the duplex and triplex in a non-cooperative manner in contrast to the cooperative binding of berberine to the DNA triplex. Strong intercalative binding to the DNA triplex structure was revealed from ferrocyanide quenching, fluorescence polarization and viscosity results. Thermal melting studies demonstrated higher stabilization of the Hoogsteen base paired third strand of the DNA triplex compared to the Watson–Crick strand. Circular dichroism studies suggested a stronger perturbation of the DNA triplex conformation by the alkaloid analogs compared to the duplex. The binding was entropy-driven in each case and the entropy contribution to free energy increased as the length of the alkyl side chain increased. The analogs exhibited stronger binding affinity to the triple helical structure compared to the parent double helical structure.     

Interpretation of DNA vibration modes: I-The guanosine and cytidine residues involved in poly(dG-dC)·poly(dG-dC) and d(CG)3·d(CG)3

Abstract

A normal coordinate analysis has been carried out on guanosine and cytidine residues appearing in oligo and polynucleotides by using a simplified valence force field that allows the vibrational spectra of 5′-dGMP and 2′-deoxycytidine molecules to be reproduced. The role of both C2′-endo and C3′-endo conformations on sugar pucker, as well as that of glycosidic torsion angle (χ), on several characteristic vibration modes of these residues have been studied. The present calculations based on a non-redundant set of internal coordinates preserving the harmonic approximation of the potential field, allows us to explain quite satisfactorily the modifications of the vibrational spectra in the 1550-1250 cm^?1 and 785-500 cm^?1 regions, when the right → left-handed conformational transition occurs.