Sanghwa Leea,b,1, Seung-Ryoung Junga,b,1, Kang Heoa,b, Jo Ann W. Bylc, Joseph E. Deweesec,d, Neil Osheroffc,e,2, and Sungchul Hohnga,f,b,2
aDepartment of Physics and Astronomy,
fBiophysics and Chemical Biology, and
bNational Center for Creative Research Initiatives, Seoul National University, Seoul 151-747, Korea;
cDepartments of Biochemistry and
eMedicine (Hematology/Oncology), Vanderbilt University School of Medicine, Nashville, TN 37232-0146; and
dDepartment of Pharmaceutical Sciences, Lipscomb University College of Pharmacy, Nashville, TN 37204-3951
Edited by Martin Gellert, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, and approved December 30, 2011 (received for review September 27, 2011)
Topoisomerase II resolves intrinsic topological problems of double-stranded DNA. As part of its essential cellular functions, the enzyme generates DNA breaks, but the regulation of this potentially dangerous process is not well understood. Here we report single-molecule fluorescence experiments that reveal a previously uncharacterized sequence of events during DNA cleavage by topoisomerase II: nonspecific DNA binding, sequence-specific DNA bending, and stochastic cleavage of DNA. We have identified unexpected structural roles of Mg2+ ions coordinated in the TOPRIM (topoisomerase-primase) domain in inducing cleavage-competent DNA bending. A break at one scissile bond dramatically stabilized DNA bending, explaining how two scission events in opposing strands can be coordinated to achieve a high probability of double-stranded cleavage. Clamping of the protein N-gate greatly enhanced the rate and degree of DNA bending, resulting in a significant stimulation of the DNA cleavage and opening reactions. Our data strongly suggest that the accurate cleavage of DNA by topoisomerase II is regulated through a tight coordination with DNA bending.
N-gate clamping, single-molecule FRET, type II topoisomerase, G-segment selection, indirect readout
1S.L. and S.-R.J. contributed equally to this work.
2To whom correspondence may be addressed.
Author contributions: S.L., S.-R.J., N.O., and S.H. designed research; S.L., S.-R.J., and K.H. performed research; J.A.W.B., J.E.D., N.O., and S.H. contributed new reagents/analytic tools; S.L., S.-R.J., and K.H. analyzed data; and S.L., S.-R.J., N.O., and S.H. wrote the paper.