title: Chiral discrimination and writhe-dependent relaxation mechanism of human topoisomerase IIα.
abstract: BACKGROUND
  Human topoisomerase IIα unlinks catenated chromosomes and preferentially relaxes positive supercoils.
  
  
  RESULTS
  Supercoil chirality, twist density, and tension determine topoisomerase IIα relaxation rate and processivity.
  
  
  CONCLUSIONS
  Strand passage rate is determined by the efficiency of transfer segment capture that is modulated by the topoisomerase C-terminal domains.
  
  
  CONCLUSIONS
  Single-molecule measurements reveal the mechanism of chiral discrimination and tension dependence of supercoil relaxation by human topoisomerase IIα. 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.
authors.affiliation: Laboratory of Molecular Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Yeonee
authors.initials: Y
authors.lastname: Seol
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Gentry
authors.forename: Neil
authors.initials: N
authors.lastname: Osheroff
authors.forename: Keir C
authors.initials: KC
authors.lastname: Neuman
mesh_terms: D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000235:genetics; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000737:chemistry* / Q000235:genetics; D004278:DNA, Superhelical / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D007536:Isomerism; D007700:Kinetics; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion; D013379:Substrate Specificity
chemical_list: D000951:Antigens, Neoplasm; D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D004250:DNA Topoisomerases, Type II
keywords: Biophysics; Chiral Discrimination; DNA Replication; DNA Topoisomerase; DNA Topology; Magnetic Tweezers; Single Molecule Biophysics