Junction between Z and B conformations in a DNA restriction fragment: evaluation by Raman spectroscopy.

Abstract

Raman vibrational spectra were obtained from two DNA restriction fragments and the DNA polymer (dGdC)n-(dG-dC)n in 0.01 and 4.5 M NaCl. One fragment contained 95 base pairs (bp) of the Eschelrichia coli lactose operator-promoter region (95-bp fragment). The other fragment consisted of the 95-bp region flanked by 26 and 32 bp ofdC-dG sequences and BamHI ends (157-bp fragment). In 0.01 M NaCl all three DNAs have Raman spectra characteristic of a right-handed B conformation. The high salt spectrum of the 95-bp fragment is also characteristic of a B conformation. However, the spectrum of the 157-bp fragment in 4.5 M NaCl shows major intensity changes from the 0.01 M NaCl spectrum. These changes are also observed in the high salt spectra of (dG-dC)n.(dG-dC)n and are correlated with the presence of a left-handed Z conformation. Comparisons between the high salt Raman spectra ofthe 157-bp fragment and spectra calculated from (dG-dC)n-(dG-dC)n and the 95-bp fragment indicated that essentially all of the dC-dG regions in the 157-bp fragment are in the Z conformation and a large part ("80%) of the 95-bp region no longer has a B-type backbone vibration. However, this non-B-DNA-like character of the central region is not indicated by base vibrations.Repeating sequences of alternating dC and dG nucleotides can form left-handed duplex DNA conformations. This was demonstrated in the solid state by x-ray diffraction studies ofcrystals of d(CpGpCpGpCpG) and d(CpGpCpG) (1-4) and fibers of the polymer (dG-dC)n-(dG-dC)n (5). The observation of left-handed conformations for dC-dG sequences helped resolve questions concerning the unusual solution properties of (dG-dC)n*(dGdC)n. It has been known for some time that spectroscopic properties of(dG-dC)n(dG-dC)n show major changes with increasing ionic strength (6). In low salt solutions (<2.0 M NaCl), circular dichroic spectra (7,8) and Raman spectra (9) are characteristic of a B-type DNA. Increased concentrations of salts or ethanol induce an inversion of the circular dichroic spectrum and changes of the Raman spectrum indicative of a cooperative intramolecular transition (8-10). The Raman spectrum of the Z conformation crystal of d(CpGpCpGpCpG) is essentially identical to the spectrum of(dG-dC)n(dG-dC)n in high salt solutions (11). This correlation together with nuclear magnetic resonance studies (12,13) indicate that the high salt form of (dG-dC)n-(dGdC)n is a Z-type conformation.Recent studies have shown that dC-dG regions can exist in a left-handed conformation when joined to a natural DNA segment. Recombinant DNA techniques were used to construct a 157-base pair (bp) restriction fragment that contains a 95-bp segment of the Escherichia coli lac operator-promoter region (14) flanked between 26 and 32 bp of dC-dG sequences (15). Fig. 1 illustrates the DNAs used in this study. The "157-bp fragment" has 153 duplex bp and the "95-bp fragment" has 95 duplex bp. Both fragments have four nucleotide tails. Circular dichroism and 31P nuclear magnetic resonance studies have shown th...

InteractionIn Vitroof Type III Intermediate Filament Proteins with Z-DNA and B-Z-DNA Junctions

“…Based on their sensitivity to S1 nuclease Singleton et al, 1984) and chemical reagents, like hydroxylamine, OsO 4 and dimethyl sulfate (Johnston and Rich, 1985;Johnston, 1992), the sites between right-handed B-DNA and left-handed Z-DNA were reported to undergo considerable structural alterations during the transition of alternating purine/pyrimidine (APP) sequences from B-to Z-DNA conformation under torsional stress. Together with the results of Raman spectroscopy studies (Wartell et al, 1982), these data proved the B-Z junctions to represent unusual, distorted DNA structures whose exact conformation is still unknown, but which is, to a certain extent, apparently single-stranded in nature (Sheardy, 1988;Lu et al, 1992). The detection in the present study of S1 nuclease-and OsO 4 /bipyridine-sensitive nucleotides at the boundaries of d(GT) 17 as well as of perfect and interrupted d(CG) 17 tracts provided an additional hint that the APP sequences had indeed adopted Z-DNA conformation, with the generation of B-Z and Z-Z junctions (Johnston et al, 1991), after their insertion into supercoiled vector DNA.…”

Unusual conformational effect exerted by Z-DNA upon its neighboring sequences.

“…For a long stretch of alternating purine/pyrimidine tracts found in human fetal globin gene intervening sequences, 20 bp or more of the flanking sequences has been reported to be sensitive to direct nuclease S1 digestion (18). Raman spectroscopy studies showed that the Z conformation can distort the structure of the neighboring region (23). On the other hand, previous chemical analyses of the B-Z junction revealed that the junctions are relatively tight, well-defined structures.…”

Characterization of nutritional parameters in bovine milk by Raman spectroscopy with least squares modeling

“…and O-C-C-N symmetric stretching -phospholipids [39] 919 X X X X* C-C stretching of α-glycosidic conformation [39,43]; NH 2 + COOH -proteic clustering [37]; C-COO stretching [43] 1006 X Ring breathing -phenylalanine and tryptophan [38,42,44] 1065 X X C-C stretching of saturated fatty acids, sensible to the acyl chain in lipids [43,45] 1086 X X C-O and C-C stretching of saccharides; C-C stretching of unsaturated fatty acid [43] 1128 X X X X C-N stretching -proteins [44]; C-O and C-C stretching of C-C 6 H 5 stretching of tryptophan and phenylalanine [38,42,44] 1249 X Amide III -deformation C-H and N-H and C-N, C-C and C-O stretching -proteins [47,48] 1266 X X Carbohydrates [39]; CH bending of oleic acid [45] 1304 X X CH 2 bending of fatty acids [43,45] 1342 X X Amide III (C-H, N-H and C-N modes) and C-C and C-O stretching -proteins [47,48]; CH deformationsaccharides (1356 cm -1 ) [39] 1438 X X CH 2 deformations -lipids [38,45] 1452 X CH 2 scissoring -proteins [47,48] 1470 X CH 2 deformation -carbohydrates [39] 1560 X X Tryptophan [36,37]; C-N stretching, NH deformation of amide II -proteins; C=C stretching of tyrosine [46,47];…”

“…acid Assignment 648 X C-C twisting -phenylalanine; C-H stretching -cysteine [36] 759 X NH 2 + COOH protein clustering -tryptophan [37] 831 X O-P-O asymmetric stretching -tyrosine [36,37] 859 X X X Tyrosine [38]; C-O-C of monosaccharide ring [39,40]; C-C-N symmetric stretching -lipids (877 cm -1 ) [41] 882 X X X X* Tryptophan aromatic ring [42] and phosphate (PO 4 ) [36];…”

“…C=C stretching of saccharides [37] 1620 X X NH 2 modes of cytosine [36,37]; β-galactose [39] 1658 X X X C=C and C=O stretching of carboxylic acids (fatty acids) [45] 1672 X C=O stretching of amide I -proteins [38] * Band with contribution to the spectrum of milk.…”