Rigidity of a B-Z region incorporated into a plasmid as monitored by electron paramagnetic resonance

Electron paramagnetic resonance spectroscopy was employed to monitor the 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 substituted in position C5 with an 11-atom-tethered 5-membered ring nitroxide (DCAVAP) was incorporated into linear (dG-dC)n 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 (Stoffel fragment). Although DCA-VAP is a modified nucleotide, it was an excellent substrate for both enzymes. The Z conformation was induced by changing 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 change was quantitatively similar for both the linear polymer and the plasmid with its Z-DNA-forming region. A motional analysis which focuses on local dynamics indicates that the order parameter S for the spin-labeled systems increases upon conversion from B-DNA to Z-DNA. This decrease in motional freedom is consistent with the observation that Z-DNA is more rigid than B-DNA.