OVERALL AND INTERNAL DYNAMICS OF DNA AS MONITORED BY 5-ATOM-TETHERED SPIN LABELS

Electron paramagnetic resonance (EPR) spectra of the two-atom-tethered six-membered ring thymidylate spin label (DUMTA) incorporated into du plexes of different sizes were found to display a helix length depende nce and a local-order parameter S = 0.32 +/- 0.01 for B-DNA based on t he dynamic cylinder model (Keyes, R. S., and A. M. Bobst. 1995. Detect ion of internal and overall dynamics of a two-atom-tethered spin-label ed DNA, Biochemistry. 34:9265-9276). This sensitivity to size, which r eflects global tumbling, is now reported for the more flexible five-at om-tethered five-membered ring thymidylate spin label (DUAP) that can be readily incorporated enzymatically and sequence specifically into n ucleic acids of different sizes. The DUAPs containing B-DNA systems we re simulated Hh the same dynamic cylinder model, giving S = 0.20 +/- 0 .01 for the more flexibly tethered spin label. This shows that S is de pendent on tether length but not on global motion. An analysis with th e same motional model of the B-Z transition in a (dG-dC)(n) polymer co ntaining the five-atom-tethered six-membered ring cytidylate spin labe l (DCAT) (Strobel, O. K., R. S. Keyes, and A. M. Bobst. 1990b. Base dy namics of local Z-DNA conformations as detected by electron paramagnet ic 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 ch anges in DNA. Both the DUAP- and the DCAT-labeled systems were also si mulated with a base disk model. From the DUAP spectral series, the per pendicular component of the correlation time tau(perpendicular to) des cribing the spin-labeled base diffusion was found to be sensitive to g lobal tumbling, confirming earlier results obtained with DUMTA. The DC AT polymer results demonstrated that tau(perpendicular to) monitors a conformational change from B- to Z-DNA, indicating that tau(perpendicu lar to) is also sensitive to local base dynamics. These results confir m that the dynamics of five-atom-tethered nitroxides are coupled to th e nucleic acid dynamics and, as with two-atom-tethered spin labels, ca n be characterized by S and tau(perpendicular to). The analyses of bot h spin-labeled systems provide good evidence for spin-labeled base mot ions within double-stranded DNA occurring on the nanosecond time scale , and establish that both labels can be used to monitor changes in glo bal tumbling and local order parameter due to variations in DNA confor mation and protein-DNA interactions.