COMPETITION BETWEEN NETROPSIN AND RESTRICTION NUCLEASE ECORI FOR DNA-BINDING

We find that netropsin and netropsin analogue protect DNA from EcoRI r estriction nuclease cleavage by inhibiting the binding of EcoRI to its recognition site. The drug - EcoRI competitive binding constants meas ured by a electrophoretic gel mobility shift assay are in excellent ag reement with the nuclease protection results for the netropsin analogu e and in reasonable agreement for netropsin itself. Crystal structures of complexes show that netropsin and EcoRI recognize different region s 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 s tructural competition with netropsin binding. The structural change in the netropsin binding region induced by EcoRI binding to its region e ssentially prevents drug association. Given the reciprocal nature of c ompetition, binding of netropsin to a minimally perturbed structure th en also makes the association of EcoRI energetically more costly. Sinc e many sequence specific DNA binding proteins significantly bend or di stort the DNA helix drugs that compete indirectly can be as effective as drugs that act through a direct steric inhibition.