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Published December 17, 2014 | public
Journal Article

Insights into copper coordination in the EcoRI-DNA complex by ESR spectroscopy

Abstract

The EcoRI restriction endonuclease requires one divalent metal ion in each of two symmetrical and identical catalytic sites to catalyse double-strand DNA cleavage. Recently, we showed that Cu^(2+) binds outside the catalytic sites to a pair of new sites at H114 in each sub-unit, and inhibits Mg^(2+)-catalysed DNA cleavage. In order to provide more detailed structural information on this new metal ion binding site, we performed W-band (similar to 94GHz) and X-band (similar to 9.5GHz) electron spin resonance spectroscopic measurements on the EcoRI-DNA-(Cu^(2+))_2 complex. Cu^(2+) binding results in two distinct components with different g_(zz) and A_(zz) values. X-band electron spin echo envelope modulation results indicate that both components arise from a Cu^(2+) coordinated to histidine. This observation is further confirmed by the hyperfine sub-level correlation results. W-band electron nuclear double resonance spectra provide evidence for equatorial coordination of water molecules to the Cu^(2+) ions.

Additional Information

© 2014 Taylor & Francis. Received 8 April 2014, accepted 9 June 2014. This work was supported by a National Science Foundation grant to Sunil Saxena and Linda Jen-Jacobson [grant number MCB 1157712; preparation of purified EcoRI and EcoRI-DNA- CU^(2-) complexes was supported by a National Institutes of Health MERIT grant to Linda Jen-Jacobson [grant number 5R37-GM029207]; the Bruker Elexsys E680 spectrometer was purchased with funds from a National Institute of Health grant [grant number 1Sl0RR028701].

Additional details

Created:
August 22, 2023
Modified:
October 19, 2023