Single-molecule measurements of the opening and closing of the DNA gate by eukaryotic topoisomerase II.
Type II DNA topoisomerases are essential and ubiquitous enzymes that perform important functions in chromosome condensation and segregation and in regulating intracellular DNA supercoiling. Topoisomerases carry out these DNA transactions by passing one segment of DNA through the other by using a reversible, enzyme-bridged double strand break. The transient enzyme/DNA adduct is mediated by a phosphodiester bond between the active-site tyrosine and a backbone phosphate of DNA. The opening and closing of the DNA gate, a critical step for strand passage during the catalytic cycle, is coupled to this cleavage/religation. We designed a unique oligonucleotide substrate with a pair of fluorophores straddling the topoisomerase II cleavage site, allowing the use of FRET to monitor the opening of the DNA gate. The DNA substrate undergoes an enzyme-mediated transition between a closed and open state in the presence of ATP, similar to the overall topoisomerase II catalyzed reaction. Single-molecule fluorescence microscopy measurements demonstrate that the transition has comparable rate constants for both the opening and closing reaction during steady-state ATP hydrolysis, with an apparent equilibrium constant near unity. In the presence of AMPPNP, a reduction in FRET occurs, suggesting an opening or partial opening of the DNA gate. However, the single-molecule experiments indicate that the open and closed states do not interconvert at a measurable rate.
Smiley
R Derike
RD
Department of Biochemistry, Duke University Medical Center, Durham, NC 27710, USA.
Collins
Tammy R L
TR
Hammes
Gordon G
GG
Hsieh
Tao-Shih
TS
D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D005057:Eukaryotic Cells / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D004247:DNA; D004250:DNA Topoisomerases, Type II