Rigidity of a B-Z Region Incorporated into a Plasmid as Monitored by Electron Paramagnetic Resonance

Author： Strobel O.K. Keyes R.S. Sinden R.R. Bobst A.M.

Publisher： Elsevier

ISSN： 0003-9861

Source： Archives of Biochemistry and Biophysics, Vol.324, Iss.2, 1995-12, pp. : 357-366

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Abstract

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 DCAVAP 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.