DNA G-segment bending is not the sole determinant of topology simplification by type II DNA topoisomerases.

Neil H Thomson,Lesley A Mitchenall,James A Taylor,Tanya Stuchinskaya,Anthony Maxwell,Sergio Santos

doi:10.1038/srep06158

Abstract

DNA topoisomerases control the topology of DNA. Type II topoisomerases exhibit topology simplification, whereby products of their reactions are simplified beyond that expected based on thermodynamic equilibrium. The molecular basis for this process is unknown, although DNA bending has been implicated. To investigate the role of bending in topology simplification, the DNA bend angles of four enzymes of different types (IIA and IIB) were measured using atomic force microscopy (AFM). The enzymes tested were Escherichia coli topo IV and yeast topo II (type IIA enzymes that exhibit topology simplification), and Methanosarcina mazei topo VI and Sulfolobus shibatae topo VI (type IIB enzymes, which do not). Bend angles were measured using the manual tangent method from topographical AFM images taken with a novel amplitude-modulated imaging mode: small amplitude small set-point (SASS), which optimises resolution for a given AFM tip size and minimises tip convolution with the sample. This gave improved accuracy and reliability and revealed that all 4 topoisomerases bend DNA by a similar amount: ~120° between the DNA entering and exiting the enzyme complex. These data indicate that DNA bending alone is insufficient to explain topology simplification and that the ‘exit gate' may be an important determinant of this process.

Highlights

DNA G-segment bending is not the sole determinant of topology simplification by type II DNA topoisomerases
Some years ago it was shown that type II DNA topoisomerases can catalyse reactions ‘beyond thermodynamic equilibrium’, that is the products of their reactions have steady-state levels of supercoiling, knotting or catenation below those seen at thermodynamic equilibrium[9]
As discussed in the Introduction, the downside of this approach is that the minimum distance of approach of the atomic force microscopy (AFM) tip to the surface is determined by the percent reduction in the free amplitude regardless of the interaction causing this reduction

Summary

Introduction

DNA G-segment bending is not the sole determinant of topology simplification by type II DNA topoisomerases. Bend angles were measured using the manual tangent method from topographical AFM images taken with a novel amplitude-modulated imaging mode: small amplitude small set-point (SASS), which optimises resolution for a given AFM tip size and minimises tip convolution with the sample This gave improved accuracy and reliability and revealed that all 4 topoisomerases bend DNA by a similar amount: ,1206 between the DNA entering and exiting the enzyme complex. The basic mechanism of all type II topoisomerases involves the passage of one double-strand segment of DNA (the ‘T’ or ‘transported’ segment) through a double-stranded break in another (the ‘G’ or ‘gate’ segment) that is stabilised by covalent linkage (phosphotyrosine bonds) with the protein[2,3] This strand-passage reaction is coupled to the binding and hydrolysis of ATP and accounts for their ability to relax and supercoil DNA and interconvert knotted/unknotted and catenated/decatenated forms[7,8]. AFM and FRET studies of three type IIA topoisomerases showed that while all three induced similar extents of G-segment bending, this did not correlate with the degree of topology simplification suggesting that G-segment bending may not be the sole determinant of topology simplification[19]

Methods

Results

Conclusion