Sequence-dependent structural features of the DNA double helix have a strong influence on the base pair opening dynamics. Here we report a detailed study of the kinetics of base pair breathing in tracts of GC base pairs in DNA duplexes derived from 1 H NMR measurements of the imino proton exchange rates upon titration with the exchange catalyst ammonia. In the limit of infinite exchange catalyst concentration, the exchange times of the guanine imino protons of the GC tracts extrapolate to much shorter base pair lifetimes than commonly observed for isolated GC base pairs. The base pair lifetimes in the GC tracts are below 5 ms for almost all of the base pairs. The unusually rapid base pair opening dynamics of GC tracts are in striking contrast to the behavior of AT tracts, where very long base pair lifetimes are observed. The implication of these findings for the structural principles governing spontaneous helix opening as well as the DNA-binding specificity of the cytosine-5-methyltransferases, where flipping of the cytosine base has been observed, are discussed.Many DNA-binding proteins are highly selective in their recognition of particular DNA sequences. Besides sequencespecific hydrogen bonding and van der Waals interactions, sequence-dependent structure and dynamics of DNA are likely to play an important role in DNA-protein interaction. In addition, the adaptability of a DNA sequence element to structural changes necessary for sequence-specific interaction is important in recognition (1).Base pair opening is required in many fundamental processes in the cell, for example, transcription and recombination. Recently, base pair opening was found to participate in a novel mode of protein-DNA interaction. The crystal structures of the M.HhaI 1 and M.HaeIII cytosine-5-methyltransferases in complex with their DNA recognition sequences showed the target base completely flipped out from the helix (2, 3). Cytosine-5-methyltransferases usually recognize a sequence of four GC base pairs (4). M.HhaI and M.HaeIII recognize 5Ј-GCGC-3Ј and 5Ј-GGCC-3Ј, respectively. A pertinent question is whether these enzymes actively expel the target base from the helix stack or capture a transient spontaneous opening. It has been shown that M.HhaI binds more tightly when a mismatch is created in the recognition sequence by replacing the target cytosine by any other base or an abasic site but not 5mC (5, 6). The enhanced binding was attributed to the lower energy required for opening a mismatched base pair upon formation of the binary complex. Hence, the base pair dynamics at the cytosine target site seems to contribute to the specificity of the cytosine-5-methyltransferases. These findings prompted us to investigate the base pair dynamics of tracts of GC base pairs.Measurements of base pair dynamics yield information about stability and structure of the double helix. Furthermore, studies of base pair opening in DNA interacting with drugs (7, 8) and hybridized with uncharged PNA (peptide nucleic acids) (9) have provided new clues to the m...

An algorithm for the study of the gradual hydration of phospholipid assemblies by means of Fourier transform infrared (FTIR) spectroscopy is presented. A complete series of diacyl phosphatidylcholines (PCs) including all possible analogues with palmitoyl and oleoyl residues, namely DPPC, DOPC, POPC, and OPPC, was investigated at room temperature. The lipid samples were prepared as cast films probably consisting of aligned multilamellar bilayers. The range of water activities studied in these films was regulated by adsorption via the gas phase corresponding to relative humidities of between 0 and 100%. Analyses of the IR‐spectroscopic data have concentrated mainly on determining the amounts of water incorporated by each lipid as well as the hydration‐induced response observed for some absorption bands of the different lipids. The water uptake at high relative humidity (RH) increases with the portion of unsaturated acyl chains in the molecular structure of the PCs. Isothermal phase transitions triggered lyotropically have been detected in demonstrating the occurrence of the main transition in POPC and OPPC films at room temperature. Moreover, it appears that both lamellar phases, the gel as well as the liquid‐crystalline phase, are not uniform. They seem to comprise an amazingly large span of order/disorder states of the lipid chains generally depending on the degree of hydration. As exemplified by the significant variation in the onset of wavenumber shifts for the PO 2− and C=O stretching‐vibration modes, obtained as a function of hydration, a sequence of attachment to polar lipid binding sites by water molecules was established for DPPC. © 1998 John Wiley & Sons, Inc. Biospectroscopy 4: 267–280, 1998

Jlilirobioloyie und experimentelle Therapie der Deutschen Akademie der Synopsis Spectrophotometric, sedimentation, infrared, optical rotatory dispersion (Oltl)), and circxilar dichroisni (CI)) methods have beeti used to demonstrate the structural changes in DNA induced by the iiiteractioii of copper(I1) with bases and to elucidate the complex binding sites. As showii by the electrolyt,e-induced reversion (addit ion of salts) of temperat~ire-deriat~ired cwpper I ) S A the effectiveness of re-formation of the doublestranded structure depends o i l the temperature, copper(I1) ioii cwiiceiitration, and on the base compositioii of the I ) S A . ICxposure of heat-deliat tired c:.)pper I)NA to higher temperatures decreases the reversioii effert on addition of e1ec.t rolyte. The reilllts indicate that a greater frartioii with a c*ooperative transitioii appwrs o i i heating I>NA to 80 or 100°C at a Cii2+/l>?;A-P ratio of 2: 1 than at a Cii2+~I)XA-1' ratio of 1 : 1 . With AT-rich copper IIXA, reveixioii t o the iiaiive I ) S A striictiire wa-; i i o t observed. Selective methylatiori of griariiiie residiies i n I)XA also afl'ects the electrol?te-itiduced reversion, itidicatiiig the importance of GC pairs for copper(I1) binding aiid the reversion to the native structure. Temperature-deriat Ilred copper 1)XA shows an increased sedimentation coefficient which decreases again after electrolyte-iiidl1c.e 1 reversion. This change in s is reduced by selective methylatioii ( J f IINA. Chrnplex formation bet ween c:opper(II) and the bases is accompaiiied by a coiiformatioiial change of the I)NA double-helical striicliire as demonstrated by O I t I ) atid CI> experiments. The O l t l ) profile of GC-rich I)NA is murh more affected by copper(I1) than that of AT-rich ones. I*;ven at very low copper(I1) coricentr:ttio~rs, e.g., at O.W aiid 0.2 Ca2+/1)NA-P, the 0111) and CU measuremeiits exhibit cotiformat ioiial changes of the DNA secondary st rrictiire at room temperattire. By comparing the itifrared spectra of deoxytiiideosides with that. of I)NA of dift'ererit (;C content it has been shown t,hat both guanine and cyt.osirie are involved i n the formation of the complex of copper(I1) with IINA. N-7 and 0 at C-6 in guanine aiid N-3 as well as 0 of C-2 in cytosine are discussed as the most probable binding sites in I)NA. A binding model for the c:oorditiation of the copper(I1) ion between giiariine arid cytosine of the opposite >traiids is sriggested. The results are in good agreement with the assumptioris :uid predictions made by Kichhorn and Clark ahout the complexirig of copper(I1) with IINA. The recent proposal made by Schreiber aiid I):iriiie :tholit :tii ititerwtioii of the type g~i : i i i i t i e -~~i 2 + -g~~: i i i i i i e canriot. he exc*ltided :is an :idditioii:il kiiid of c~ooi~diii:itioii o f (*oppw(Il) i t 1 1)X.k.Various divaleiit metal ions are kno\vii to interact nit11 riucleic acids.L-LO Iri some investigations the results of metal determination in carefully isolated D S A preparations iiidicat e that certain metal io...

The structure of iremycin [10-(alpha-L-rhodosaminyl)-gamma-rhodomycinone] hydrochloride has been confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. We studied the interaction of iremycin and the related compound daunomycin with DNA by transient electric dichroism and by sedimentation analysis of supercoiled closed duplex DNA. The apparent length increase of sonicated calf thymus DNA (150 +/- 20 base pairs) in 2.5 mM sodium cacodylate buffer (pH 7) at 12 degrees C was determined to be 0.40 +/- 0.02 nm/bound drug). The Cu(II) complex of iremycin with a metal/drug ratio of 0.7 induces a length increase of DNA of 0.44 +/- 0.02 nm/added drug. The alignment of the iremycin chromophore with respect to the DNA helix axis was determined from the electric dichroism of the complex. The tilt (long axis) and twist (short axis) of the chromophore are both 28 +/- 4 degrees, whereas for daunomycin the long axis is perpendicular to the helix axis and the short axis is twisted by about 25 degrees. Intercalation of iremycin between DNA base pairs is supported by unwinding of the supercoiled closed duplex form of pBR 322 plasmid DNA from Escherichia coli. In 2.5 mM sodium cacodylate buffer at pH 7 and at 25 degrees C, the unwinding induced by iremycin is 15.0 +/- 1.5 degrees/bound drug. Under identical conditions daunomycin shows on unwinding angle of 15.4 +/- 1.5 degrees. The superhelical density of pBR 322 DNA (sigma 0) was determined to be -0.087 +/- 0.002 at standard conditions (0.2 M NaCl, 37 degrees C).