No abstract

We find that netropsin and netropsin analogue protect DNA from EcorI restriction nuclease cleavage by inhibiting the binding of EcoRI to its recognition site. The drug -- EcoRI competitive binding constants measured by a electrophoretic gel mobility shift assay are in excellent agreement with the nuclease protection results for the netropsin analogue and in reasonable agreement for netropsin itself. Crystal structures of complexes show that netropsin and EcoRI recognize different regions of the DNA helix and would not be expected to compete for binding to the restriction nuclease site. The large distortions in DNA structure caused by EcoRI binding are most likely responsible for an indirect structural competition with netropsin binding. The structural change in the netropsin binding region induced by EcoRI binding to its region essentially prevents drug association. Given the reciprocal nature of competition, binding of netropsin to a minimally perturbed structure then also makes the association of EcoRI energetically more costly. Since many sequence specific DNA binding proteins significantly bend or distort the DNA helix, drugs that compete indirectly can be as effective as drugs that act through a direct steric inhibition.

Previous electric birefringence experiments have shown that the actin-activated Mg2+-ATPase activity of Acanthamoeba myosin II correlates with the ability of minifilaments to cycle between flexible and stiff conformations. The cooperative transition between conformations was shown to depend on Mg2+ concentration, on ATP binding, and on the state of phosphorylation of three serines in the C-terminal end of the heavy chains. Since the junction between the heavy meromyosin (HMM) and light meromyosin (LMM) regions is expected to disrupt the alpha-helical coiled-coil structure of the rod, this region was anticipated to be the flexible site. We have now cloned and expressed the wild-type rod (residues 849-1509 of the full-length heavy chain) and rods mutated within the junction in order to test this. The sedimentation and electric birefringence properties of minifilaments formed by rods and by native myosin II are strikingly similar. In particular, the Mg2+-dependent flexible-to-stiff transitions of native myosin II and wild-type rod minifilaments are virtually superimposable. Mutations within the junction between the HMM and LMM regions of the rod modulate the ability of Mg2+ to stabilize the stiff conformation. Less Mg2+ is required to induce minifilament stiffening if proline-1244 is replaced with alanine. Deleting the entire junction region (25 amino acids) results in a even greater decrease in the Mg2+ concentration necessary for the transition. The HMM-LMM junction does indeed seem to act as a Mg2+-dependent flexible hinge.