Investigation of DNA structural changes by infrared spectroscopy

The structure of DNA in aqueous trifluoroethanol solutions containing more than 70% (vol/vol) trifluoroethanol, where it has been proposed to have the A-conformation, has been studied by the technique of electric linear dichroism. The data demonstrate that both linear 4X174DNA [5386 base pairs (bp) long] and a 172-bp fragment of sea urchin SS RNA gene are in the double-helical A-form in the alcoholic solution and that two of the important structural parameters, the base tilt and the base-pair translation along the helical axis, are the same as in the A-form structure of crystalline double-helical d(GGTATACC). Despite its molecular length, the limiting reduced dichroism (-1.41) of the 4X174 DNA in low-salt aqueous solution is near the theoretical limit (-1.50) for DNA with base planes perpendicular to the helical axis. Transformation of this DNA and also of the 172-bp fragment to the A-structure in alcohol solutions reduces the experimental value of the dichroism to about -1.04, which agrees almost exactly with the value of -1.03 calculated for the A-form crystal. The base-pair translation along the helical axis was determined from relaxation of the DNA dichroism when the electric field is turned off. The 172-bp fragment is found to have a base-pair translation ofonly 2.6 A in the A-structure and is thus the same as in A-DNA fibers and in the A-form oligomers crystals. We conclude from the coincidence of the base-pair translation and the apparent values of the base tilt that the A structures are identical or nearly so in the solution and solid states. Comparison of the 4X174 data with data on smaller DNA fragments and with the data on the 172-bp sea urchin gene fragment unambiguously confirms that, in aqueous solution, the experimental reduced electric dichroism ofB-DNA is highly dependent on molecular length, while the dichroism of the A-DNA form in alcoholic solutions is independent of molecular length at least in the range from 172 to 5386 bp. Values of the optical dichroism associated with known absorption bands in these substances are intrinsically related to their structural parameters. Using the technique of electric linear dichroism, two studies (9-11) of DNA in aqueous alcoholic solution have, however, resulted in entirely divergent observations with respect to the values of the Oichroism of the A-structure. Wu et al. (9), using small fragments [180 base pairs (bp)] of enzymatically cleaved pBR322, observed that the linear dichroism became more negative in alcoholic solutions under conditions where the A-structure is expected to predominate, while Lee and Charney (10, 11), using sonicated calf-thymus DNA with mean lengths of about 250 and 1300 bp, observed that the dichroism becomes less negative (than in aqueous nonalcoholic solutions) under similar conditions. In the latter study, unsonicated "native" calf thymus DNA appeared to aggregate under the same conditions, and no significance could be attributed to the measured dichroism of this high molecular weight sample in alcoholic solution. I...

mentioning

confidence: 88%

Structure of A-DNA in solution.

Charney¹,

Chen²

1987

“…Although both the SASP-DNA complex and the DNA control give a maximum in the 1185-cm-1 band at about 70% relative humidity, the latter loses intensity much more rapidly with further decrease in the humidity. Such observations on bands attributable to DNA conformation (secondary structure) have been interpreted to mean that the molecule has become "disordered" (29). DISCUSSION CD changes have long been regarded as sensitive criteria for DNA conformations (21)(22)(23)(24) and, though it may be difficult to assign fine structural details on the basis of CD spectra, the distinctions between the main families of secondary structure are unambiguous.…”

Section: Figurementioning

confidence: 99%

Binding of small acid-soluble spore proteins from Bacillus subtilis changes the conformation of DNA from B to A.

Mohr

Sokolov²,

He³

et al. 1991

132

108

Small acid-soluble spore proteins (SASPs) appear 3-4 hr after the onset of sporulation in Gram-positive bacteria and constitute up to 20% of the protein of mature spores. Previous studies using Bacillus subtlis deletion mutants lacking SASP-a and -1S have shown that such mutations abolish the elevated resistance ofspores to UV radiation. Analyses using circular dichroism and Fourier-transform infrared spectroscopy now demonstrate that bining a/f-type SASPs to DNA in vitro causes a structural change in DNA, from the B to the A conformation. This may provide the basis whereby a/f8-type SASPs confer increased spore UV resistance in vivo-by changing spore DNA conformation, they alter DNA photochemistry such that UV irradiation produces spore photoproduct instead of the more lethal cyclobutane-type thymine dimers.genes (termed ssp genes) exhibit a remarkably high degree of conservation of their primary sequences throughout evolution (10), and any one ofthe a/a-type SASPs appears capable ofconferring UV resistance on bacterial spores (11). By using several purified a/,a-type SASPs, we have examined the effects of SASP on DNA conformation, and we report here that SASP binding to DNA in vitro induces a B -> A conformation change. It seems likely that this phenomenon underlies the ability of SASP to protect spore DNA against thymine-dimer formation. Given their evolutionary antiquity (10), the SASPs also may have as-yet-undiscovered protein relatives that use the same strategy to protect cells against UV. The high intrinsic UV resistance of obligate anaerobic bacteria (12), for example, may reflect just such a system. Among the dramatic changes accompanying sporulation in Gram-positive bacteria, a major increase in resistance to killing by UV radiation ranks high as a factor ensuring that the organism will survive dormancy (1). Detailed study of this phenomenon has shown that UV irradiation of spores produces no detectable cis-syn cyclobutane-type thymine dimers in DNA-in sharp contrast with the predominance of this lesion in other UV-irradiated in vivo systems. Instead, the DNA of UV-irradiated spores accumulates primarily 5-thyminyl-5,6-dihydrothymine ("spore photoproduct") (2,3). Germinating spores contain a specific enzyme that efficiently repairs spore photoproduct (4), thereby ensuring a high probability of survival after exposure to UV.It is clear that DNA conformation can significantly alter photoreactivity (5) and the type of UV-induced photoproducts (6). Thus, one simple explanation for the change in DNA photochemistry that occurs during sporulation invokes an alteration in DNA conformation. Indeed, to explain the altered photochemistry of spore DNA, it was suggested a number of years ago that DNA in bacterial spores is in the A conformation whose stereochemical constraints inhibit thymine dimer formation (7). Howe-, er, the relationship between A-DNA and UV-induced formation of spore photoproduct has never been proven, and it has been suggested (6, 8) that hydration is a more important determinant of D...

“…were prepared for CD measurements by first carefully spreading the polymer solution on quartz plates in such a manner as to avoid orientation and then placing them in a specially designed humidity chamber that resembled those used for infrared measurements (12,13).…”

mentioning

confidence: 99%

“…Beckman IR 9 spectrophotometer equipped with a wire grid polarizer by a previously described procedure (13).…”

mentioning

confidence: 99%

Nature of conformational changes in poly[d(A-T)-d(A-T)] in the premelting region.

Brahms¹,

Brahms²,

Holde³

1976

The conformation of the synthetic DNA, poly-[d(A-T)d(A T)J, has been investigated both in the solid state and in dilute aqueous solutions at different temr ratures below its melting point The change of the circular dichroism (CD) spectra of poly[d(A-T)d(A-T)J solutions with decreasing temperatures from just below the melting point to 00 involves a specific de-crease of the intensity of the 262 nm CD band. This conformational change has been assigned to a gradual and partial transition from the B to C form, on the basis of the following results:(i) By the use of infrared dichroism measurements on oriented films we have defined humidity and salt conditions under which One of the unexplained phenomena in molecular biology is the exact nature of premelting changes in DNAs. Many observations indicate the occurrence of significant changes in DNA structure at temperatures below the helix-coil transition. Such changes were initially detected by optical rotation at the sodium D line (1) and by viscosity measurements (2). They have since been confirmed by the more sensitive method of UV circular dichroism (3). That these occur in a temperature region below the helix-coil transition is indicated by the constant absorbance at 260 nm. The positive circular dichroism (CD) band of DNA becomes more intense as the temperature increases toward the melting temperature (tm). Explanations proposed for the premelting phenomenon postulate the existence of some conformational alterations of DNA before the transition to the random coil (3). More recently these CD observations have been confirmed and extended to different DNAs of various origins and base compositions (4). All DNAs exhibit the CD premelting phenomenon. Oligomers of d(AT)n with n = 10-21 yielded results similar to those found for the polymer, without showing chain length dependence. This indicates that the cooperative unit in this transition is very small (4).Although the premelting phenomenon in DNA can be detected by several methods, it is not observable by changes in hypochromicity (3, 4) or by Raman scattering (5, 6). In fact, investigation of poly[d(A-T)-d(A-T)] by laser Raman scattering indicates the absence of any premelting spectral changes (7), whereas in DNA the changes before melting are observed only at temperature higher than about 50°C (5).Since the premelting changes of DNA are observed by CD at temperatures of biological reactions, they may be important in biological processes. For example, they may be relevant for the understanding of interactions of DNA with proteins as diverse as histones, polymerases, or repressors. In order to make precise structural determinations we have chosen to investigate poly[d(A-T)-d(A-T)] for which different A, B, and C forms, as well as a particular D form, have been well characterized by x-ray diffraction (8, 9) and by infrared linear dichroism (10). We have used CD to investigate both films and solutions in order to establish structural correlations between the two states. MATERIALS AND METHODS Samples of poly[d(A-...