Chiral Discrimination and Writhe-dependent Relaxation Mechanism of Human Topoisomerase IIα

Abstract

Type IIA topoisomerases (Topo IIA) are essential enzymes that relax DNA supercoils and remove links joining replicated chromosomes. Human topoisomerase IIα (htopo IIα), one of two human isoforms, preferentially relaxes positive supercoils, a feature shared with Escherichia coli topoisomerase IV (Topo IV). The mechanistic basis of this chiral discrimination remains unresolved. To address this important issue, we measured the relaxation of individual supercoiled and “braided” DNA molecules by htopo IIα using a magnetic tweezers-based single-molecule assay. Our study confirmed the chiral discrimination activity of htopo IIα and revealed that the strand passage rate depends on DNA twist, tension on the DNA, and the C-terminal domain (CTD). Similar to Topo IV, chiral discrimination by htopo IIα results from chiral interactions of the CTDs with DNA writhe. In contrast to Topo IV, however, these interactions lead to chiral differences in relaxation rate rather than processivity. Increasing tension or twist disrupts the CTD-DNA interactions with a subsequent loss of chiral discrimination. Together, these results suggest that transfer segment (T-segment) capture is the rate-limiting step in the strand passage cycle. We propose a model for T-segment capture that provides a mechanistic basis for chiral discrimination and provides a coherent explanation for the effects of DNA twist and tension on eukaryotic type IIA topoisomerases. Type IIA topoisomerases (Topo IIA) are essential enzymes that relax DNA supercoils and remove links joining replicated chromosomes. Human topoisomerase IIα (htopo IIα), one of two human isoforms, preferentially relaxes positive supercoils, a feature shared with Escherichia coli topoisomerase IV (Topo IV). The mechanistic basis of this chiral discrimination remains unresolved. To address this important issue, we measured the relaxation of individual supercoiled and “braided” DNA molecules by htopo IIα using a magnetic tweezers-based single-molecule assay. Our study confirmed the chiral discrimination activity of htopo IIα and revealed that the strand passage rate depends on DNA twist, tension on the DNA, and the C-terminal domain (CTD). Similar to Topo IV, chiral discrimination by htopo IIα results from chiral interactions of the CTDs with DNA writhe. In contrast to Topo IV, however, these interactions lead to chiral differences in relaxation rate rather than processivity. Increasing tension or twist disrupts the CTD-DNA interactions with a subsequent loss of chiral discrimination. Together, these results suggest that transfer segment (T-segment) capture is the rate-limiting step in the strand passage cycle. We propose a model for T-segment capture that provides a mechanistic basis for chiral discrimination and provides a coherent explanation for the effects of DNA twist and tension on eukaryotic type IIA topoisomerases.