Torsion and bending of nucleic acids studied by subnanosecond time-resolved fluorescence depolarization of intercalated dyes

Millar, David P.; Robbins, Rebecca J.; Zewail, Ahmed H.

doi:10.1063/1.443182

Cited by 158 publications

(122 citation statements)

References 70 publications

(56 reference statements)

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“…It is interesting that 5Z shows a single slow decay (Ϸ5 ns); 5G shows a similar decay but with an additional decay component (Ϸ12%) of Ϸ100 ps. The Ϸ100-ps and Ϸ5-ns anisotropy decays of 5G are ascribed to the restricted rotation of E in DNA and other slower rotations, including that of the whole DNA duplex (37,39,40). As discussed elsewhere (34), the fact that the Ϸ100-ps anisotropy decay exists in 5G but not in 5Z indicates that there is a correlation of the slow ET process (75 ps) with the rotation of E in DNA.…”

Section: Et Dynamicsmentioning

confidence: 77%

Femtosecond dynamics of DNA-mediated electron transfer

Wan

Fiebig

Kelley

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

373

362

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Diverse biophysical and biochemical studies have sought to understand electron transfer (ET) in DNA in part because of its importance to DNA damage and its repair. However, the dynamics and mechanisms of the elementary processes of ET in this medium are not fully understood and have been heavily debated. Two fundamental issues are the distance over which charge is transported and the time-scale on which the transport through the -stack of the DNA base pairs may occur. With femtosecond resolution, we report direct observation in DNA of ultrafast ET, initiated by excitation of tethered ethidium (E), the intercalated electron acceptor (A); the electron donor (D) is 7-deazaguanine (Z), a modified base, placed at different, fixed distances from A. The ultrafast ET between these reactants in DNA has been observed with time constants of 5 ps and 75 ps and was found to be essentially independent of the D-A separation (10-17 Å). However, the ET efficiency does depend on the D-A distance. The 5-ps decay corresponds to direct ET observed from 7-deazaguanine but not guanine to E. From measurements of orientation anisotropies, we conclude that the slower 75-ps process requires the reorientation of E before ET, similar to E͞nucleotide complexes in water. These results reveal the nature of ultrafast ET and its mechanism: in DNA, ET cannot be described as in proteins simply by a phenomenological parameter, ␤. Instead, the involvement of the base pairs controls the time scale and the degree of coherent transport.The striking resemblance of the base-pair stack of DNA to conductive one-dimensional aromatic crystals prompted, over 30 years ago, the proposal that long-range charge transport might proceed through DNA (1). In the three decades since, biochemical, biophysical, and theoretical studies have sought to address the possibility and efficiency of the transport (2-29). Such charge migration through DNA is significant, because radical migration is a critical issue to our understanding of carcinogenesis and mutagenesis (5, 6).Photoinduced electron transfer (ET) reactions have provided a useful tool in elucidating parameters governing ET through DNA. In the 1980s and early 90s, a class of experiments on noncovalently bound electron donors (D) and electron acceptors (A) in DNA was reported (7-12). A major debate focused on whether or not ET through DNA may proceed rapidly and differently from that found in -bonded systems. These experiments provided valuable information and raised many questions, but a key issue was the distance between D and A, which was not well defined.With D and A covalently bonded to DNA, studies of ET on more well defined assemblies were made possible, and the effect of distance could be addressed (13)(14)(15)(16)(17)(18)(19)(20). Values of the parameter ␤, which reflects the distance scale of ET through a given medium (30), remarkably, ranged from Յ0.1 Å Ϫ1 to Ͼ1.4 Å Ϫ1; ␤ was estimated by using one system of a fixed distance (13,14) or by varying the distance (15-20). The fact that values of ␤ coul...

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Section: Et Dynamicsmentioning

confidence: 77%

Femtosecond dynamics of DNA-mediated electron transfer

Wan

Fiebig

Kelley

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

373

362

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“…Structural nuclear magnetic resonance analysis of an A-hmU base pair flanked by G-C base pairs indicates no significant deviations from classical B-form DNA (21), although nearest neighbors do influence the structure (22). 13 C or 31 P nuclear magnetic resonance relaxation experiments may provide the required insights into the dynamics of duplex DNA the size of a protein binding site, features that are only poorly extractable from measurements using techniques such as fluorescence polarization and DNA cyclization (23,24). The suggested locations of DNA bends in the TF1⅐DNA complex coincide with two hmU-A base pair steps.…”

Section: Resultsmentioning

confidence: 99%

Twin Hydroxymethyluracil-A Base Pair Steps Define the Binding Site for the DNA-bending Protein TF1

Grove

Figueiredo

Galeone

et al. 1997

Journal of Biological Chemistry

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The DNA-bending protein TF1 is the Bacillus subtilis bacteriophage SPO1-encoded homolog of the bacterial HU proteins and the Escherichia coli integration host factor. We recently proposed that TF1, which binds with high affinity (K d was ϳ3 nM) to preferred sites within the hydroxymethyluracil (hmU)-containing phage genome, identifies its binding sites based on sequence-dependent DNA flexibility. Here, we show that two hmU-A base pair steps coinciding with two previously proposed sites of DNA distortion are critical for complex formation. The affinity of TF1 is reduced 10-fold when both of these hmU-A base pair steps are replaced with A-hmU, G-C, or C-G steps; only modest changes in affinity result when substitutions are made at other base pairs of the TF1 binding site. Replacement of all hmU residues with thymine decreases the affinity of TF1 greatly; remarkably, the high affinity is restored when the two hmU-A base pair steps corresponding to previously suggested sites of distortion are reintroduced into otherwise T-containing DNA. T-DNA constructs with 3-base bulges spaced apart by 9 base pairs of duplex also generate nM affinity of TF1. We suggest that twin hmU-A base pair steps located at the proposed sites of distortion are key to target site selection by TF1 and that recognition is based largely, if not entirely, on sequence-dependent DNA flexibility.The genome of the Bacillus subtilis bacteriophage SPO1 contains 5-hydroxymethyluracil (hmU) 1 in place of thymine, and discrimination between T-and hmU-containing DNA is important during phage multiplication (1, 2). For one phageencoded protein, the DNA-binding and -bending protein TF1, this discrimination is essentially absolute: the high affinity of TF1 (K d order of magnitude nM) for preferred sites within the hmU-containing phage genome is greatly reduced for the corresponding T-containing DNA (3, 4). We recently showed that the affinity of TF1 for hmU-DNA is matched in T-containing DNA constructs that contain sets of two consecutive mismatches separated by 9 bp of duplex, indicating that hmUcontent and appropriately placed flexible loops contribute similarly to complex formation. We proposed, therefore, that DNA in a complex with TF1 is distorted at two sites that are separated by 9 bp of duplex DNA, sites corresponding to two hmU-A base pair steps, and that TF1 identifies its target sites through recognition of sequence-dependent DNA flexibility (5). Here we examine the requirement for hmU residues across a 37-mer DNA duplex representing a preferred binding site within the phage genome (4) and specifically evaluate sequence requirements at the two inferred sites of DNA distortion. EXPERIMENTAL PROCEDURESTF1 was overexpressed in Escherichia coli DL39 (6) and purified as described (5). Oligonucleotides with hmU content were synthesized as described (7) and purified by high pressure liquid chromatography. T-containing oligonucleotides were purchased and purified by denaturing polyacrylamide gel electrophoresis. The top strand (shared among bulge constructs...

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“…The intercalated probe has different nanosecond fluorescence properties from those of E in water, and one can distinguish between intercalated and nonintercalated E molecules (1)(2)(3)(4)(5). Time-resolved studies of E intercalated in DNA on the picosecond time scale provide a probe of torsional dynamics and DNA flexibility, which are important structural features of DNA (7)(8)(9)(10).…”

mentioning

confidence: 99%

Femtosecond dynamics of the DNA intercalator ethidium and electron transfer with mononucleotides in water

Fiebig

Wan

Kelley

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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Ethidium (E) is a powerful probe of DNA dynamics and DNA-mediated electron transfer (ET). Molecular dynamical processes, such as solvation and orientation, are important on the time scale of ET. Here, we report studies of the femtosecond and picosecond time-resolved dynamics of E, E with 2deoxyguanosine triphosphate (GTP) in water, and E with 7-deaza-2-deoxyguanosine triphosphate (ZTP) in water; E undergoes ET with ZTP but not GTP. These studies elucidate the critical role of relative orientational motions of the donor-acceptor complex on ET processes in solution. For ET from ZTP to E, such motions are in fact the ratedetermining step. Our results indicate that these complexes reorient before ET. The time scale for the solvation of E in water is 1 ps, and the orientational relaxation time of E is 70 ps. The impact of orientational and solvation effects on ET between E and mononucleotides must be considered in the application of E as a probe of DNA ET.Because of its unique properties, ethidium (E) bromide has been used widely for various biochemical and biophysical applications (e.g., refs. 1-10). The cationic dye E intercalates in helical polynucleotides and can be used as a nucleic acid stain. The intercalated probe has different nanosecond fluorescence properties from those of E in water, and one can distinguish between intercalated and nonintercalated E molecules (1-5). Time-resolved studies of E intercalated in DNA on the picosecond time scale provide a probe of torsional dynamics and DNA flexibility, which are important structural features of DNA (7-10).E has also been employed as a probe of DNA-mediated electron transfer (ET) reactions (11-16). Recently, it has been shown, in systems employing E covalently tethered to DNA duplexes, that on photoexcitation this intercalator acts as an electron acceptor with respect to a modified guanine base, 7-deazaguanine (11), or as a donor with respect to a rhodium intercalator (12). In these studies, measurements of fluorescence quenching suggested fast reaction kinetics (Ն10 10 s Ϫ1) and a sensitivity of quenching to stacking.Despite the general utility of E as a DNA probe, its photophysical properties on the ultrafast time scale have not been examined extensively. Sommer et al. (17) measured the transient fluorescence spectra with picosecond time resolution and observed a time-dependent spectral shift on a subnanosecond time scale in glycerol. This shift was attributed to an intramolecular relaxation process involving phenyl-group rotation. The fact that they could not resolve the dynamics of this process when water was used as a solvent has been attributed to the lower viscosity, which offers no significant hindrance to phenyl-group rotation.Studies of the femtosecond dynamics of E in water and ET processes of E with mononucleotides lay the foundation for studies of ultrafast ET dynamics in DNA and time-resolved studies of biological dynamics. Here, we report our studies of the femtosecond dynamics of E and of ET between E and mononucleotides in water. Expe...

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Torsion and bending of nucleic acids studied by subnanosecond time-resolved fluorescence depolarization of intercalated dyes

Cited by 158 publications

References 70 publications

Femtosecond dynamics of DNA-mediated electron transfer

Femtosecond dynamics of DNA-mediated electron transfer

Twin Hydroxymethyluracil-A Base Pair Steps Define the Binding Site for the DNA-bending Protein TF1

Femtosecond dynamics of the DNA intercalator ethidium and electron transfer with mononucleotides in water

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