Single-molecule measurements of the opening and closing of the DNA gate by eukaryotic topoisomerase II

@article{citeulike:1717602, abstract = {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. 10.1073/pnas.0700342104}, author = {Smiley, Derike R. and Collins, Tammy R. and Hammes, Gordon G. and Hsieh, Tao-Shih }, citeulike-article-id = {1717602}, doi = {10.1073/pnas.0700342104}, journal = {Proceedings of the National Academy of Sciences}, keywords = {fret}, month = {March}, number = {12}, pages = {4840--4845}, posted-at = {2007-10-02 01:21:19}, priority = {2}, title = {Single-molecule measurements of the opening and closing of the DNA gate by eukaryotic topoisomerase II}, url = {http://dx.doi.org/10.1073/pnas.0700342104}, volume = {104}, year = {2007} }

TY - JOUR ID - citeulike:1717602 L3 - citeulike-article-id:1717602 TI - Single-molecule measurements of the opening and closing of the DNA gate by eukaryotic topoisomerase II JF - Proceedings of the National Academy of Sciences VL - 104 IS - 12 SP - 4840 EP - 4845 N2 - 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. 10.1073/pnas.0700342104 KW - fret AU - Smiley, Derike AU - Collins, Tammy AU - Hammes, Gordon AU - Hsieh, Tao-Shih PY - 2007/03/20/ UR - http://dx.doi.org/10.1073/pnas.0700342104 ER -