High affinity DNA-microtubule associated protein interaction

Abstract

We have isolated the MAP/tau proteins from twice-cycled chick brain microtubule preparations and demonstrated that they are responsible for the nitrocellulose DNA binding activity we and others have measured. Using the isolated MAP/tau proteins we then measured the apparent affinity constant K_app for the homologous chick DNA interaction and found evidence for two equilibrium affinity classes-a K_app = 6 × 10⁷ M⁻¹, responsible for the bulk of the DNA binding activity and a small (< 10%) higher affinity K_app = 10⁸ − 10⁹ M⁻¹, likely due to sequence specific binding protein species. Using the same chick brain MAP-tau protein, a heterologous interaction with D. melanogaster DNA, was found to possess just the lower affinity class-K_app = 2 × 10⁷ M⁻¹. Under stringent binding conditions we carried out equilibrium nitrocellulose filter binding experiments in a ternary reaction mixture at constant MAP/tau protein and ³⁵S radiolabelled chick DNA concentration using increasing and excess concentrations of competitor DNAs of different sources. The order of competitor strengths found was-chick DNA > mouse DNA > D. melanogaster = E. coli. DNA. These data and specifically the homologous DNA: protein case being the strongest competitor corroborate our previous studies using total microtubule protein and provide new evidence for a conserved interaction of a small DNA sequence class with MAP/tau protein species. Moreover, these data allow us to conclude that the conserved DNA sequence: MAP/tau protein interactions do not critically depend upon any energetic feature co-involving tubulin for their properties since tubulin is absent from these preparations.