Spin-Labeled Nucleotide Mobility in the Boundary of the EcoRI Endonuclease Binding Site

Abstract

A complex consisting of the EcoRI endonuclease site-specifically bound to spin-labeled DNA 26mers was prepared to provide a model system for studying possible conformational changes resulting from protein binding. EPR was used to monitor the mobility of the spin labels that were strategically placed in position 6, 9, or 11 with respect to the dyad axis of the 26mer. These positions are located within the flanking region on either side of the EcoRI hexamer binding site. This allows the monitoring of potential distal structural changes in the DNA helix caused by protein binding. The spectral line shapes indicate that the spin label closest to the EcoRI endonuclease binding site, i.e., in position 6, is most influenced by the binding event. The EPR data are analyzed according to a model that distinguishes between spectral effects due to a change in the hydrodynamic shape of the complex and those resulting from local variations in the spin-label mobility as characterized by a local order parameter S. S reflecting the motional restriction of the spin-labeled base is 0.20 ± 0.01 for all three oligomers as well as for the two complexes with the label in position 9 or 11, while the position 6 labeled complex yields S=0.25. To further evaluate the origin of the slightly larger EPR effect observed with position 6 labeled material, molecular dynamics (MD) simulations were used to explore the space accessible to the probes in positions 6, 9, and 11. MD results gave similar nitroxide trajectories for all three labeled 26mers in the absence or presence of EcoRI. Thus, the small position 6 effect is attributed to a structural distortion in the major groove of the DNA at this location possibly corresponding to a bend induced by protein binding. The observation that the spectral changes are small indicates the absence of any significant structural disruption being propagated along the helix as a result of protein binding. Also, the fact that the line shape of the 26mers did not change as expected from hydrodynamic theory in view of the significant increase in molecular volume upon protein binding suggests that there are additional relaxation processes involving the protein and nucleic acid.     

The universal ancestor and the ancestors of Archaea and Bacteria were anaerobes whereas the ancestor of the Eukarya domain was an aerobe

The use of an oxyphobic index (OI) based on the propensity of amino acids to enter more frequently the proteins of anaerobes makes it possible to make inferences on the environment in which the last universal common ancestor (LUCA) lived. The reconstruction of the ancestral sequences of proteins using a method based on maximum likelihood and their attribution by means of the OI to the set of aerobe or anaerobe sequences has led to the following conclusions: the LUCA was an anaerobic 'organism', as were the ancestors of Archaea and Bacteria, whereas the ancestor of Eukarya was an aerobe. These observations seem to falsify the hypothesis that the LUCA was an aerobe and help to identify better the environment in which the first organisms lived.