RIGIDITY OF A B-Z REGION INCORPORATED INTO A PLASMID AS MONITORED BY ELECTRON-PARAMAGNETIC-RESONANCE

Electron paramagnetic resonance spectroscopy was employed to monitor t he dynamics associated with a B-Z transition in both a linear (dG-dC)( n) and a modified pUC8 plasmid. A spin label consisting of cytidine su bstituted in position C5 with an 11-atom-tethered 5-membered ring nitr oxide (DCAVAP) was incorporated into linear (dG-dC), with Micrococcus luteus DNA polymerase or into a specific 34-bp Z-DNA-forming region of the 2.7-kb plasmid pRDZ8 with Thermus aquaticus DNA polymerase (Stoff el fragment). Although DCA-VAP is a modified nucleotide, it was an exc ellent substrate for both enzymes. The Z conformation was induced by c hanging the salt concentration from 0.01 to 4.5 M. The EPR line shape changed in response to the switch in DNA conformation. The degree of c hange was quantitatively similar for both the linear polymer and the p lasmid with its Z-DNA-forming region. A motional analysis which focuse s on local dynamics indicates that the order parameter S for the spin- labeled systems increases upon conversion from B-DNA to Z-DNA. This de crease in motional freedom is consistent with the observation that Z-D NA is more rigid than B-DNA. (C) 1995 Academic Press, Inc.