Dipsticking the major groove of DNA with enzymatically incorporated spin-labeled deoxyuridines by electron spin resonance spectroscopy

Bobst, Albert M.; Kao, Shih-Chung; Toppin, Roland C.; Ireland, John C.; Thomas, Ingrid E.

doi:10.1016/0022-2836(84)90403-0

Cited by 45 publications

(11 citation statements)

References 18 publications

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“…As the tether length increases, the value of 11 for six-membered ring nitroxides decreases approximately linearly. The observation that both six-and eleven-atom tethers are well described by Ti = 0.06 ns can be explained by earlier results indicating that the spin-label nitroxide ring is moved beyond the major groove into the bulk environment upon changing the tether length from five to six atoms (Bobst et al, 1984). It seems that beyond a tether length of six atoms, 1 remains nearly constant.…”

Section: Consistency Of Simulation Parametersmentioning

confidence: 60%

Overall and internal dynamics of DNA as monitored by five-atom-tethered spin labels

Keyes

Bobst

Cao

et al. 1997

Biophysical Journal

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Electron paramagnetic resonance (EPR) spectra of the two-atom-tethered six-membered ring thymidylate spin label (DUMTA) incorporated into duplexes of different sizes were found to display a helix length dependence and a local-order parameter S = 0.32 +/- 0.01 for B-DNA based on the dynamic cylinder model (Keyes, R. S., and A. M. Bobst. 1995. Detection of internal and overall dynamics of a two-atom-tethered spin-labeled DNA. Biochemistry. 34:9265-9276). This sensitivity to size, which reflects global tumbling, is now reported for the more flexible five-atom-tethered five-membered ring thymidylate spin label (DUAP) that can be readily incorporated enzymatically and sequence specifically into nucleic acids of different sizes. The DUAPs containing B-DNA systems were simulated with the same dynamic cylinder model, giving S = 0.20 +/- 0.01 for the more flexibly tethered spin label. This shows that S is dependent on tether length but not on global motion. An analysis with the same motional model of the B-Z transition in a (dG-dC)n polymer containing the five-atom-tethered six-membered ring cytidylate spin label (DCAT) (Strobel, O. K., R. S. Keyes, and A. M. Bobst. 1990b. Base dynamics of local Z-DNA conformations as detected by electron paramagnetic resonance with spin-labeled deoxycytidine analogues. Biochemistry. 29:8522-8528) revealed an increase in S from 0.15 +/- 0.01 to 0.26 +/- 0.01 in response to the B- to Z-DNA transition. This indicates that S is not only sensitive to tether length, but also to conformational changes in DNA. Both the DUAP- and the DCAT-labeled systems were also simulated with a base disk model. From the DUAP spectral series, the perpendicular component of the correlation time tau perpendicular describing the spin-labeled base diffusion was found to be sensitive to global tumbling, confirming earlier results obtained with DUMTA. The DCAT polymer results demonstrated that tau perpendicular monitors a conformational change from B- to Z-DNA, indicating that tau perpendicular is also sensitive to local base dynamics. These results confirm that the dynamics of five-atom-tethered nitroxides are coupled to the nucleic acid dynamics and, as with two-atom-tethered spin labels, can be characterized by S and tau perpendicular. The analyses of both spin-labeled systems provide good evidence for spin-labeled base motions within double-stranded DNA occurring on the nanosecond time scale, and establish that both labels can be used to monitor changes in global tumbling and local order parameter due to variations in DNA conformation and protein-DNA interactions.

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Section: Consistency Of Simulation Parametersmentioning

confidence: 60%

Overall and internal dynamics of DNA as monitored by five-atom-tethered spin labels

Keyes

Bobst

Cao

et al. 1997

Biophysical Journal

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“…Site-specific covalent spin-labeling methods for nucleic acids have been carried out in some selected tRNAs containing minor bases in positions known from sequencing (Bobst, 1979). Direct attachment of a spin-labeled probe to DNA has been accomplished using both enzymatic and chemical syntheses (Bobst et al, 1984;Kao & Bobst, 1985;Pauly et al, 1987;Spaltenstein et al, 1989;Kirchner et al, 1990). It is clear from these studies that the length and structure of the tether which connects the nitroxide reporter group to the DNA have a profound influence on the EPR spectra obtained.…”

mentioning

confidence: 99%

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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“…The nature of the tether ensures that there is only one degree of motional freedom of the probe relative to the base and thus minimizes independent probe motion. Bobst and co-workers (Bobst et al, 1984(Bobst et al, , 1988; Kao & Bobst, 1985;Pauly et al, 1987;Strobel et al, 1990) have developed an alternative set of spin-labels for studying DNA dynamics with longer, less rigid tethers. They analyze their data in terms of anisotropic rotational diffusion with one correlation time dependent on tether length and ascribed to independent probe motion, while the second correlation time is independent of tether and helix length (Bobst et al, 1984) and attributed to motion of the base.…”

mentioning

confidence: 99%

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

Hustedt

Spaltenstein²,

Kirchner³

et al. 1993

Biochemistry

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The dynamics of a series of four DNA duplexes of length 12, 24, 48, and 96 base pairs have been studied using an electron paramagnetic resonance (EPR) active nitroxide spin-label covalently attached to a thymidine located near the center of each duplex. The linear EPR spectra were simulated by solving the stochastic Liouville equation for anisotropic rotational diffusion. The diffusion tensor for global rotation of the duplex was predicted from hydrodynamic theory for a right circular cylinder. All internal motions, assumed to be rapid, are modeled by reduced electron Zeeman and hyperfine tensor anisotropies. Best fit simulations to the data were then obtained by adjusting the total amplitude of all internal dynamics. The local, length-independent and the collective, length-dependent contributions to the internal dynamics were separated by determining the total amplitude of internal motion as a function of duplex length. The major axis of the spin tensors was determined to be tilted 20 degrees from the helix axis. As a result, the spin-label is most sensitive to flexural motions of the DNA duplex. It is found that the global tumbling of duplex is accurately modeled by hydrodynamic theory. The length-independent motion is characterized by a root-mean-squared amplitude of oscillation of 10 degrees in two dimensions at 20 degrees C and has a strong temperature dependence, indicating that the local structure of the DNA changes with temperature. The length dependence of the internal dynamics leads to an estimate of the dynamic flexural persistence length of 2500 +/- 340 A. There was no statistically significant difference between models assuming a harmonic or a square-well local bending potential.

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Dipsticking the major groove of DNA with enzymatically incorporated spin-labeled deoxyuridines by electron spin resonance spectroscopy

Cited by 45 publications

References 18 publications

Overall and internal dynamics of DNA as monitored by five-atom-tethered spin labels

Overall and internal dynamics of DNA as monitored by five-atom-tethered spin labels

Spin-Labeled Psoralen Probes for the Study of DNA Dynamics

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

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