Studying the Differences in Relaxation Activity by Topoisomerase I and III at the Single Molecule Level

Abstract

E. coli topoisomerases I and III (Topo I and Topo III) can relax negatively (-) supercoiled DNA, and also catenate or decatenate DNA molecules containing single-stranded DNA regions. Although these enzymes share the same mechanism of activity and have similar structures, they participate in different cellular processes: Topo I is mainly involved in transcription whereas Topo III is involved in recombination, reflected in bulk experiments as a more efficient DNA relaxation activity by Topo I and a more efficient catenation activity by Topo III (1,2). This raises the question: what characteristics of Topo I and Topo III are responsible for their different behavior?To examine the differences in the activity mechanisms of the two related type IA topoisomerases, single molecule relaxation studies were conducted on several DNA substrates: (-) supercoiled DNA, (+) supercoiled DNA with a 12bp/ 27bp mismatch, and (+) supercoiled DNA with a 12bp/ 27bp bulge. Three major differences between the mechanism of Topo I and Topo III were examined: the time lag before initiation of relaxation events, the rates of DNA relaxation, and the total rates of DNA relaxations. The experiments show differences in the way the two proteins work at the single molecule level, while also recovering the bulk experiments observations. The results provide insights into the mechanism of both proteins, help to understand their differences, and explain why Topo I is more efficient than Topo III in relaxing (-) supercoiled DNA.1. Tse-Dinh, Y. C., Bacterial and archeal type I topoisomerases. Biochim Biophys Acta, 1998. 1400(1–3): p. 19–27.2. Zhu, Q., P. Pongpech, and R. J. DiGate, Type I topoisomerase activity is required for proper chromosomal segregation in Escherichia coli. Proc. Natl. Acad. Sci. USA, 2001. 98(17): p. 9766–71.