Topoisomerase II resolves intrinsic topological problems of doublestranded 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 singlemolecule 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 2þ 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.he double helical nature of DNA imposes intrinsic topological problems during replication, repair, and transcription (1-3). Additionally, the topological state of the genetic material needs to be tightly regulated in order to promote proper biochemical interactions between DNA and a variety of proteins (1-4). Topoisomerases are enzymes that resolve topological problems within the double helix by repeated cycles of DNA cleavage and ligation (1-3, 5, 6).As a subclass of the topoisomerase family, type II topoisomerases are found in all organisms from bacteria to human, and even in some viruses (1-3, 5, 6). The essential roles of type II topoisomerases in cell metabolism, differences between bacterial and human homologues, and hyperactivation of these enzymes in cancer cells have been utilized for clinical treatments of bacterial infections and numerous cancers (3, 7-9).Extensive studies for more than twenty years have established that type II topoisomerases use a "two gate" mechanism for DNA strand passage (3, 10, 11), in which a DNA duplex (the transport or T-segment) is transported through an enzyme-mediated transient opening in a separate DNA duplex (the gate or G-segment). The directionality of strand passage is the N-terminal gate of the enzyme to the C-terminal gate. As a result of the double-stranded DNA passage mechanism, each catalytic event changes the linking number of DNA by two. The transport of the T-segment through the G-segment is thought to be initiated by the N-gate clamping motion induced by the binding of ATP to the enzyme (3,6,10,12).Although the double-stranded DNA breaks generated by type II topoisomerases are essential for the cellular functions of these enzymes, it is a dangerous process in which an aberrant operation can damage chromosomal integrity. In fact, widely prescribed anticancer and antibacterial drugs initiate cell death by increasing the cellular conce...
We developed a hybrid technique combining optical tweezers and single-molecule three-color fluorescence resonance energy transfer (FRET). In demonstrative experiments, we observed the force-sensitive correlated motion of three helical arms of a Holliday junction and identified the independent unfolding/folding dynamics of two DNA hairpins of the same length. With 3 times the number of observable elements of single-molecule FRET, this new instrument will enable the measurement of the complex, multidimensional effects of mechanical forces in various biomolecular systems, such as RNA and proteins.
In neuronal exocytosis, SNARE assembly into a stable four-helix bundle drives membrane fusion. Previous studies have revealed that the SM protein Munc18-1 plays a critical role for precise SNARE assembly with the help of Munc13-1, but the underlying mechanism remains unclear. Here, we used single-molecule FRET assays with a nanodisc membrane reconstitution system to investigate the conformational dynamics of SNARE/Munc18-1 complexes in multiple intermediate steps towards the SNARE complex. We found that single Munc18-1 proteins induce the closed conformation of syntaxin-1 not only in the free syntaxin-1 but also in the t-SNARE (syntaxin-1/SNAP-25) complex. These results implicate that Munc18-1 may act as a gatekeeper for both binary and ternary SNARE complex formation by locking the syntaxin-1 in a cleft of Munc18-1. Furthermore, the kinetic analysis of the opening/closing transition reveals that the closed syntaxin-1 in the syntaxin-1/SNAP-25/ Munc18-1 complex is less stable than that in the closed syntaxin-1/Munc18-1 complex, which is manifested by the infrequent closing transition, indicating that the conformational equilibrium of the ternary complex is biased toward the open conformation of syntaxin-1 compared with the binary complex. Neuronal exocytosis for neurotransmitter release is driven by the assembly of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are syntaxin-1 and SNAP-25 in the presynaptic plasma membrane and synaptobrevin in the synaptic vesicle membrane 1,2. The assembly of these proteins generates a stable four-helix bundle between the vesicle and target membranes, thus promoting membrane fusion 3,4. Although the membrane fusion can be induced by the SNAREs alone in vitro, a number of auxiliary proteins are required for membrane fusion with high speed and high fidelity in vivo 5,6. Among those, the Sec1/Munc18 (SM) family proteins Munc18-1 and Munc13-1 are known to be essential for SNARE-mediated membrane fusion 7-9. Extensive studies on roles of the Munc18-1 and Munc13-1 in the membrane fusion have established that these proteins are critically involved in the regulation of SNARE complex formation. Initially, Munc18-1 induces the closed conformation of syntaxin-1, locking the syntaxin-1 protein in a cleft of Munc18-1, that inhibits the spontaneous binding of SNAP-25 to syntaxin-1 10,11. Recent reports have revealed that the MUN domain of Munc13-1 promotes the transition from the syntaxin-1/Munc18-1 complex to the ternary SNARE complex in the presence of SNAP-25 and synaptobrevin, suggesting that Munc13-1 plays a role in opening syntaxin-1 for the subsequent SNARE assembly 12-15. On the other hand, it is also known that Munc18-1 stimulates membrane fusion when it binds to a fully assembled SNARE complex 16-18. Despite such major advances, many important questions concerning the mechanisms underlying the precise regulation of SNARE complex formation by the Munc18-1 and Munc13-1 still remain unanswered. For instance,
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