Unknotting by Type II Topoisomerases

One intriguing family of knots is the group of twist knots; they are interesting from both a knot theory perspective and a biochemical perspective. These knots exist with any number of crossings, but their unknotting number is one! A twist knot consists of an interwound (or twisted) region with any number of crossings and a clasp with two crossings. The critical crossing lies within this clasp. By reversing one of the crossings in the clasp the twist knot is converted to the unknot.

Unknotting a Twist Knot

When the topology of a DNA molecule is a twist knot, the interwound region corresponds to supercoils and the clasp results from strand passage of two distant segments of the DNA molecule. The unknotting number of a mathematical knot K is analogous to the smallest number of topoisomerase strand passage events needed to untie a DNA knot.

Topoisomerases are enzymes that pass one DNA strand through another via an enzyme-bridged transient break in the DNA. Topoisomerases act locally on DNA to solve problems of global DNA entanglement. Type II topoisomerases reduce/induce supercoiling in DNA molecules and are capable of cleavage, passage and ligation action on double-stranded DNA. The specific mechanism that allows type II topoisomerases to unknot and decatenate DNA in vivo is as yet unknown, although various models have been proposed. We are interested in the topological species controlled by type II topoisomerases. By combining theory, simulation and experiment we hope to gain more insight into the mechanism of these enzymes.

Currently I am doing experimental work in the lab of E. Lynn Zechiedrich at Baylor College of Medicine, Houston, TX. This project is being done in collaboration with my FSU advisor De Witt Sumners and, at Baylor, Lynn Zechiedrich and Rick Deibler. The goal of this project is to investigate the unknotting activity of type II topoisomerases. We have purified preparative amounts of several types of DNA knots and developed a novel approach to topoisomerase unknotting experiments.

One specific aim is to determine if a type II topoisomerase will unknot a DNA twist knot in one cycle of action. To address this aim we are investigating human topoisomerase IIα unknotting activity on 5- and 7-noded DNA twist knots. A single crossing change in the clasp region of the knot produces the unknot. However, a crossing change in the twisted region produces a twist knot with two less nodes. Preliminary data indicates that human topoisomerase IIα unknots DNA twist knots by acting upon a crossing in the clasp region of the knot. In particular, the 5-noded DNA twist knots acted upon by human topoisomerase IIα are converted to the unknot with no appearance of the trefoil, a potential product if the enzyme acts on a crossing within the twist region. Additionally, the 7-noded DNA twist knots acted upon by human topoisomerase IIα are also converted to the unknot with no appearance of either the trefoil or the 5-noded twist knot, both of which are potential intermediates if the enzyme acts on crossings within the twist region. Our results indicate that human topoisomerase IIα converts 3-, 5- and 7-noded DNA twist knots to the unknot.

This picture was taken while I was attending the workshop “Statistical Mechanics of Polymer Models” at Banff International Research Station, Alberta, Canada.