Stivers J T, Harris T K, Mildvan A S
Center for Advanced Research in Biotechnology of the University of Maryland Biotechnology Institute and National Institute of Standards and Technology, Rockville 20850, USA.
Biochemistry. 1997 Apr 29;36(17):5212-22. doi: 10.1021/bi962880t.
The Vaccinia type I topoisomerase catalyzes site-specific DNA strand cleavage and religation by forming a transient phosphotyrosyl linkage between the DNA and Tyr-274, resulting in the release of DNA supercoils. For type I topoisomerases, two mechanisms have been proposed for supercoil release: (I) a coupled mechanism termed strand passage, in which a single supercoil is removed per cleavage/religation cycle, resulting in multiple topoisomer intermediates and late product formation, or (2) an uncoupled mechanism termed free rotation, where multiple supercoils are removed per cleavage/religation cycle, resulting in few intermediates and early product formation. To determine the mechanism, single-turnover experiments were done with supercoiled plasmid DNA under conditions in which the topoisomerase cleaves predominantly at a single site per DNA molecule. The concentrations of supercoiled substrate, intermediate topoisomers, and relaxed product vs time were measured by fluorescence imaging, and the rate constants for their interconversion were determined by kinetic simulation. Few intermediates and early product formation were observed. From these data, the rate constants for cleavage (0.3 s(-1)), religation (4 s(-1)), and the cleavage equilibrium constant on the enzyme (0.075) at 22 degrees C are in reasonable agreement with those obtained with small oligonucleotide substrates, while the rotation rate of the cleaved DNA strand is fast (approximately 20 rotations/s). Thus, the average number of supercoils removed for each cleavage event greatly exceeds unity (delta n = 5) and depends on kinetic competition between religation and supercoil release, establishing a free rotation mechanism. This free rotation mechanism for a type I topoisomerase differs from the strand passage mechanism proposed for the type II enzymes.
痘苗I型拓扑异构酶通过在DNA与Tyr-274之间形成瞬时磷酸酪氨酸连接来催化位点特异性DNA链的切割和重新连接，从而导致DNA超螺旋的释放。对于I型拓扑异构酶，已提出两种超螺旋释放机制：（1）一种称为链穿通的偶联机制，其中每个切割/重新连接循环去除一个超螺旋，导致多个拓扑异构中间体和晚期产物形成；或（2）一种称为自由旋转的非偶联机制，其中每个切割/重新连接循环去除多个超螺旋，导致少量中间体和早期产物形成。为了确定该机制，在拓扑异构酶主要在每个DNA分子的单个位点切割的条件下，用超螺旋质粒DNA进行了单周转实验。通过荧光成像测量超螺旋底物、中间拓扑异构体和松弛产物的浓度随时间的变化，并通过动力学模拟确定它们相互转化的速率常数。观察到少量中间体和早期产物形成。根据这些数据，在22℃下切割（0.3 s⁻¹）、重新连接（4 s⁻¹）的速率常数以及酶上的切割平衡常数（0.075）与用小寡核苷酸底物获得的值合理一致，而切割的DNA链的旋转速率很快（约20转/秒）。因此，每个切割事件去除的超螺旋平均数量大大超过1（Δn = 5），并且取决于重新连接和超螺旋释放之间的动力学竞争，从而确立了自由旋转机制。这种I型拓扑异构酶的自由旋转机制不同于为II型酶提出的链穿通机制。
