THE VISCOELASTIC PROPERTIES OF DNA; EFFECT OF A TOPOLOGY CONTROLLING ENZYME AND ITS TARGETING INHIBITORS

Abstract

The flow properties of DNA are important for understanding cell division and, indirectly, cancer therapy. DNA topology controlling enzymes such as topoisomerase II are thought to play an essential role.

The viscoelastic moduli of phage Lambda DNA through the entanglement transition were obtained with particle tracking microrheology. With increasing frequency, the viscous loss modulus first increases, then levels off, and eventually increases again. Concurrently, the elastic storage modulus monotonously increases and eventually levels off to a constant high frequency plateau value. Once the DNA molecules become entangled at about ten times the overlap concentration, the elastic storage modulus becomes larger than the viscous loss modulus in an intermediate frequency range. The number of entanglements per chain is obtained from the plateau value of the elasticity modulus. The longest, global relaxation time pertaining to the motion of the DNA molecules is obtained from the low shear viscosity as well as from the lowest crossover frequency of the viscous loss and elastic storage moduli. The concentration dependencies of the low shear viscosity, the number of entanglements per chain, and the relaxation time agree with the relevant scaling laws for reptation dynamics of entangled polyelectrolytes in an excess of simple, low molecular weight salt with screened electrostatic interactions.

This methodology is used to show how double strand passage facilitated by topoisomerase II controls DNA rheology. For this purpose, elastic storage and viscous loss moduli of a model system comprising bacteriophage Lambda DNA and human topoisomerase II alpha are measured using video tracking of the Brownian motion of colloidal probe particles. It is found that the rheology is critically dependent on the formation of temporal entanglements among the DNA molecules with a relaxation time on the order of a second. It is observed that topoisomerase II effectively removes these entanglements and transforms the solution from an elastic physical gel to a viscous fluid depending on the consumption of adenosine-triphosphate (ATP). Another aspect of study is the effect of the generic topoisomerase II inhibitor adenylyl-imidodiphosphate (AMP-PNP) and the anticancer drug bisdioxopiperazine ICRF-193. In mixtures of AMP-PNP and ATP, the double strand passage reaction gets blocked and progressively fewer entanglements are relaxed. A total replacement of ATP by AMP-PNP results in a temporal increase in elasticity at higher frequencies, but no transition to an elastic gel with fixed cross-links is observed. Addition of anticancer drugs results in the inhibition of topoisomerase II.