DNA cleavage and opening reactions of human topoisomerase IIα are regulated via Mg2+-mediated dynamic bending of gate-DNA.
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 Mg(2+) 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.
Department of Physics and Astronomy, Seoul National University, Seoul 151-747, Korea.
Sanghwa
S
Lee
Seung-Ryoung
SR
Jung
Kang
K
Heo
Jo Ann W
JA
Byl
Joseph E
JE
Deweese
Neil
N
Osheroff
Sungchul
S
Hohng
D024342:Amino Acids, Acidic / Q000378:metabolism; D000951:Antigens, Neoplasm / Q000378:metabolism*; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D053837:DNA Cleavage / Q000187:drug effects*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008274:Magnesium / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation / Q000187:drug effects*
D024342:Amino Acids, Acidic; D000951:Antigens, Neoplasm; D002413:Cations, Divalent; D004268:DNA-Binding Proteins; D004247:DNA; D004250:DNA Topoisomerases, Type II; D008274:Magnesium