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

Abstract

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.

A Spin-Labeled Abasic DNA Substrate for AP Endonuclease

“…Our high-field results are consistent with previous estimates of local ordering based on low-frequency ESR of dsDNA labeled with a 5-atom flexible tether (14,15). At lower ESR frequencies, it is necessary to account for both the global tumbling of the DNA and the local motion of the label using the sophisticated Slowly Relaxing Local Structure (SRLS) model (16).…”

Structure and dynamics of a DNA-based model system for the study of electron spin–spin interactions

“…Paramagnetic manganese can substitute for magnesium in some catalytic RNAs [14][15][16][17][18] and a wide variety of spin-labeled nucleotides have been developed. [5,6,[19][20][21][22][23][24] The interactions of paramagnetic lanthanide ions with nucleic acids, directly or indirectly, are also of interest. The substitution of Er(III) for Mg(II) inhibits Dicer, a specialized ribonuclease while the substitution of La(III) for Mg(II) improves the processivity of reverse transcriptase, a DNA polymerase.…”

Site-directed Spin Labeling Studies on Nucleic Acid Structure and Dynamics

“…Data on B-form DNA duplexes were simulated using a model that characterizes nitroxide motions by two correlation times, τ ‖ and τ ⊥ (Section V.B.2) (25,29,101,109). τ ‖ was found to be tether dependent, was less than 1 ns, and was attributable to nitroxide motion that was independent of base motion (11).…”

“…τ ⊥ was thought to represent motions including twisting and tilting of the individual base, collective bending and twisting of the DNA segment, and global tumbling of DNA (25,29,101,109). Values of τ ⊥ from all double-stranded systems were reported to be in the 1-10 ns range (11,25,29,101,109), leading to the conclusion that there are significant base motions and these motions contribute to the dynamics of the nitroxide. However, questions have been raised as to the degree to which τ ⊥ is influenced by the motion of the tether versus that of the base (see below) (14).…”

“…There has been debate over whether the rigidly coupled nitroxides alter the behavior of the base, and if so, to what degree (11,14,29,101,109). On the other hand, for the flexible nitroxides, questions have been raised regarding whether motions of the flexible linker lead to an overestimation of base dynamics (14).…”