We presented an X-band pulse EPR spectrometer with high throughput and excellent sensitivity in the 8.5-11.5GHz range [1] and illustrated the capabilities and performance of the spectrometer by measurements on free radicals and biradicals in solids and liquids. The advantages of direct detection of highly stable deuterated nitroxides and the simplicity of the measurements of electron spin relaxation times will be demonstrated.
During the last years we applied CW and pulse dipolar EPR to study complexes of DNA with enzymes responsible for DNA reparation. Pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology [2]. During the last years we used NMR [3] and EPR [4] to study structure of complexes of DNA with enzymes. In particular we adapted the CLEANEX-PM NMR protocol to detect DNA imino protons exchange and analyzed the dynamics of oxoG:C, oxoG:A and their undamaged counterparts in nucleotide contexts with different stacking energy. Even in a poorly stacking context, the oxoG:C pair did not open easier than G:C, arguing against extrahelical base capture by Fpg/OGG1.
To understand the role of ionizable groups in the catalytic mechanism of repair enzymes we synthesized DNA oligonucleotides spin-labeled at the 5'-end of DNA with spin tags based on imidazoline and imidazolidine nitroxyl radicals sensitive to pH/electrostatics and proximity of water molecules, using nitroxyl radicals. EPR spectra at different pH were measured for spin-labeled DNA duplexes and for duplex-enzyme complexes. The availability of water in the active center was investigated by ESEEM and ENDOR.
This work was supported by Russian Science Foundation № 21-14-00219.
References:
[1] N.P. Isaev, et al., JMRO, 2023, 14-15, 100092.
[2] O.Schiemann,et al. JACS. 2021, 143, 43, 17875-17890.
[3] S. Ovcherenko, et al. JACS.2023, 145, 10, 5613–5617
[4] O.A. Krumkacheva, et al. NAR, 2019, V. 47, N 15, Pp 7767-7780