Motions of short DNA duplexes: An analysis of DNA dynamics using an EPR-active probe

Hustedt, Eric J.; Spaltenstein, Andrew; Kirchner, James J.; Hopkins, Paul B.; Robinson, Bruce H.

doi:10.1021/bi00058a011

Cited by 61 publications

(98 citation statements)

References 49 publications

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“…Steric inhibition of rotation of the nitroxide by the surrounding DNA has been implicated as the factor that allows such long correlation times to be observed (Kirchner et al, 1990). A significant amplitude of bending in addition to the overall anisotropic rigid body motion was found for a 46 bp duplex labeled with this probe (Hustedt et al, 1993(Hustedt et al, , 1995.…”

mentioning

confidence: 98%

Spin-Labeled Psoralen Probes for the Study of DNA Dynamics

Hp¹,

Dy²,

Ng³

et al. 1995

Biochemistry

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Six nitroxide spin-labeled psoralen derivatives have been synthesized and evaluated as probes for structural and dynamic studies. Sequence specific photoaddition of these derivatives to DNA oligonucleotides resulted in site-specifically cross-linked and spin-labeled oligomers. Comparison of the general line shape features of the observed electron paramagnetic resonance (EPR) spectra of several duplexes ranging in size from 8 to 46 base pairs with simulated EPR spectra indicate that the nitroxide spin-label probe reports the global tumbling motion of the oligomers. While there is no apparent large amplitude motion of the psoralen other than the overall tumbling of the DNA on the time scales investigated, there are indications of bending and other residual motions. The (A)BC excinuclease DNA repair system detects structural or dynamic features of the DNA that distinguish between damaged and undamaged DNA and are independent of the intrinsic structure of the lesion. NMR studies have shown that psoralencross-linked DNA has altered backbone dynamics and conformational populations in the immediate vicinity Psoralen-damaged DNA serves as an excellent substrate for the study of structural and dynamic motifs that DNA repair enzyme systems may recognize. Psoralens are photoreagents which form a well-characterized set of covalent nucleic acid adducts through photochemical addition ( Figure 1) (Straub et al., 1981; Kanne et al., 1982a,b). The primary reaction is cyclobutane ring formation between the 5,6 double bond of thymidine in DNA and either the 4',5' or 3,4 double bonds of the psoralen. Reaction at the 4'3' double bond creates a furanside monoadduct (MAf),' which can react further at a site with opposed and adjacent pyrimidines to create an interstrand cross-link (XL). The XL is a rigid unit formed by the psoralen and the opposite and adjacent pyrimidine bases, linking the two strands together. Reaction at the 3,4 double bond of the psoralen first creates a pyroneside monoadduct (MAP), which cannot form an interstrand cross-link.Psoralen-DNA monoadducts and cross-links are recognized and removed by the excision repair system in both

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confidence: 98%

Spin-Labeled Psoralen Probes for the Study of DNA Dynamics

Hp¹,

Dy²,

Ng³

et al. 1995

Biochemistry

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“…This report demonstrates that MP substitution does increase the flexibility and that the increased flexural root-mean-square (rms) bending is comparable to the mean bend induced by the MP substitution. The entropic and electrostatic contributions attributable to MP substitution are also investigated.Site-specific duplex DNA flexibility and differential flexibility attributable to changes in structure have not been experimentally quantified before largely because of technical limitations.Continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy (16,17), in combination with a site-specific base pair, composed of 2-aminopurine (P) and a spin-labeled nitroxide quinolone (Q) (18)(19)(20), is used to investigate the submicrosecond flexibility of duplex DNA with and without MP substitution. The probe is part of the sixth base pair (P⅐Q) in the duplex and monitors the dynamics of the DNA at this position.…”

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confidence: 99%

“…Continuous-wave electron paramagnetic resonance (CW-EPR) spectroscopy (16,17), in combination with a site-specific base pair, composed of 2-aminopurine (P) and a spin-labeled nitroxide quinolone (Q) (18)(19)(20), is used to investigate the submicrosecond flexibility of duplex DNA with and without MP substitution. The probe is part of the sixth base pair (P⅐Q) in the duplex and monitors the dynamics of the DNA at this position.…”

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confidence: 99%

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Phosphate backbone neutralization increases duplex DNA flexibility: A model for protein binding

Okonogi

Alley

Harwood

et al. 2002

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

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An important component of protein-DNA recognition is the charge neutralization of DNA backbone phosphates and subsequent protein-induced DNA bending. Replacement of phosphates by neutral methylphosphonates has previously been shown to be a model for protein-induced bending. In addition to bending, the neutralization process may change the inherent flexibility of the DNA-a feature never before tested. We have developed a method to measure the differential flexibility of duplex DNA when methylphosphonate substitutions are made and find that the local flexibility is increased up to 40%. These results imply that backbone-neutralization-dependent DNA flexibility augments DNAbinding motifs in protein-DNA recognition processes.electrostatics ͉ DNA bending ͉ methylphosphonate ͉ phantom proteins T wenty years ago, Mirzabekov and Rich suggested that neutralization of the phosphate backbone of duplex DNA by cationic amino acids contributed to the bending and flexibility of DNA bound to histones (1). Recently this idea has been tested by using model systems in which specific phosphates on DNA have been neutralized (2-4) by replacing phosphate groups with methylphosphonates (MPs). It was shown that strategically placed runs or patches of MPs induced bends of 3-4°per base pair in DNA. The magnitude of bends induced in DNA upon binding positively charged mutants of GCN4, a bZIP transcription factor, compared favorably with the bends generated in the unbound DNA by substituting equal numbers of MPs for phosphates adjacent to the basic region in GCN4 (5). These and other examples (5-7) illustrate the significant contribution of chargeneutralization-induced DNA bending to the binding energetics of protein recognition processes.Increased DNA flexibility could contribute to the energetics of protein-DNA binding by allowing the DNA to accommodate a greater degree of bending and a wider range of structural conformations at the same energy. Transient electric birefringence studies of meroduplexes (in which one side of the duplex DNA has no sugar-phosphate backbone and no charge) found no bends between base pairs (6°per base pair is the lower detection limit of the experiment) but found a suggestion of increased flexibility (8). Theoretical estimates indicate that the electrostatic contribution to the static bending ranges from negligible (9-11) to about a 5°bend per base pair (12). Theoretical estimates of the electrostatic contribution to the dynamic bending also range from increased flexibility (1, 2) through a negligible effect on flexibility (13, 14) to reduced flexibility (15), heightening the importance of experimentally testing whether MP-modified DNA actually alters the internal flexibility of duplex DNA. This report demonstrates that MP substitution does increase the flexibility and that the increased flexural root-mean-square (rms) bending is comparable to the mean bend induced by the MP substitution. The entropic and electrostatic contributions attributable to MP substitution are also investigated.Site-specific duplex DNA f...

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“…Hence, measurements in such systems provide information on the average behavior of the duplex rather than site-specific data. In order to examine properties of duplex DNA as a function of sequence and position, great effort has been expended in developing probes that can be covalently bound to specific sites for use in EPR 10,[17][18][19][20][21][22][23] and in labeling specific atoms by isotopic substitution to prepare sequences for NMR studies. 10,[24][25][26][27] EPR labeling experiments have focused on replacing natural bases with analogs, modified to contain the EPR active nitroxide radical as an integral part of the base.…”

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confidence: 99%

Theory for Spin−Lattice Relaxation of Spin Probes on Weakly Deformable DNA

et al. 2008

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The weakly bending rod (WBR) model of double-stranded DNA (dsDNA) is adapted to analyze the internal dynamics of dsDNA as observed in electron paramagnetic resonance (EPR) measurements of the spin-lattice relaxation rate, R 1e , for spin probes rigidly attached to nucleic acid-bases. The WBR theory developed in this work models dsDNA base-pairs as diffusing rigid cylindrical discs connected by bending and twisting springs whose elastic force constants are κ and R, respectively. Angular correlation functions for both rotational displacement and velocity are developed in detail so as to compute values for R 1e due to four relaxation mechanisms: the chemical shift anisotropy (CSA), the electron-nuclear dipolar (END), the spin rotation (SR), and the generalized spin diffusion (GSD) relaxation processes. Measured spin-lattice relaxation rates in dsDNA under 50 bp in length are much faster than those calculated for the same DNAs modeled as rigid rods. The simplest way to account for this difference is by allowing for internal flexibility in models of DNA. Because of this discrepancy, we derive expressions for the spectral densities due to CSA, END, and SR mechanisms directly from a weakly bending rod model for DNA. Special emphasis in this development is given to the SR mechanism because of the lack of such detail in previous treatments. The theory developed in this paper provides a framework for computing relaxation rates from the WBR model to compare with magnetic resonance relaxation data and to ascertain the twisting and bending force constants that characterize DNA.

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Motions of short DNA duplexes: An analysis of DNA dynamics using an EPR-active probe

Cited by 61 publications

References 49 publications

Spin-Labeled Psoralen Probes for the Study of DNA Dynamics

Spin-Labeled Psoralen Probes for the Study of DNA Dynamics

Phosphate backbone neutralization increases duplex DNA flexibility: A model for protein binding

Theory for Spin−Lattice Relaxation of Spin Probes on Weakly Deformable DNA

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