High affinity DNA-microtubule interactions: evidence for a conserved DNA-MAP interaction involving unusual high CsCl density repetitious DNA families

Abstract

We have examined high affinity interactions of chick brain microtubule proteins with ³⁵S labelled tracer DNAs from chick, mouse and D. melanogaster under equilibrium conditions by the nitrocellulose filter binding technique. Ternary reaction mixtures of the above two components and a third component, an excess of unlabelled competitor DNA from either E. coli., mouse, D. melanogaster or chick, were used to measure small fractions of DNA in each case (1–4%) bound to microtubule protein under high stringency- large competitor DNA concentration and 0.5 M NaCl. As seen in part previously (Marx, K.A. and Denial, T. (1985) in The Molecular Basis of Cancer, 172B, 65–75 (Rein, ed), A. Liss, N.Y.) the measured order of competitor DNA strengths was identical for all three tracer DNAs. That is: chick > mouse > D. melanogaster > E. coli competitor DNA. Since the homologous interaction, chick competitor DNA with chick brain microtubule protein, is always the strongest interaction measured, we interpret this as evidence for a conserved protein-DNA sequence interaction. ³⁵S chick DNA tracer sequences, isolated from nitrocellulose filters following the stringent binding in the presence of 0.9 mM⁻¹ E. coli. competitor DNA, was used in driven reassociation reactions with total chick driver DNA. This fraction was found to be significantly enriched in repetitive chick DNA sequences. Since we have observed a similar phenomenon in mouse, we then compared the stringent binding mouse sequences and showed that the bulk of these sequences did not cross-hybridize with total chick DNA. Finally, all three ³⁵S tracer DNAs binding to nitrocellulose were isolated and sedimented to equilibrium on CsCl density gradients. The CsCl density distributions from all three DNAs showed significant (100-fold) enrichment in classical satellite DNAs as well as higher enrichment in two very unusual high CsCl density families of DNA (1.720–1.740 g/cm³; 1.750–1.765 g/cm³). These families are never observed as distinct bands in total DNA CsCl gradients, nor could we isolate them in purified tubulin control binding experiments. This apparently general phenomena may be identifying some of the sequence families involved in the high affinity microtubule interaction, which appears to be conserved in evolution.