Direct observation of strand passage by DNA-topoisomerase and its limited processivity.
Direct observation of strand passage by DNA-topoisomerase and its limited processivity.
复制标题
DOI：
10.1371/journal.pone.0034920
复制
发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Kinosita K Jr
中科院分区：
文献类型：
--
作者：
Yogo K;Ogawa T;Hayashi M;Harada Y;Nishizaka T;Kinosita K Jr
ii型DNA拓扑异构酶通过将DNA双链的一个片段通过另一个片段的瞬时酶桥联双链断裂来解决DNA缠结，如超螺旋、结和链链。先前在单分子水平上已经证明了依赖ATP的通道反应，在饱和ATP时显示出明显的进程性。在这里，我们用光学镊子将一对荧光染色的DNA分子缠绕30圈后，通过人拓扑异构酶i α直接观察了链的通过。平均有0.51±0.33µm（11±6匝）的编织链在8±4 s的反应中断开，断开长度基本上与0.25-37 pM之间的酶浓度无关。过程解联开始前的时间随着酶浓度的减少而减少，在3.7 pM时为~ 100 s。这些结果与酶与一个DNA结合约10秒的情况一致，等待与另一个DNA的多次扩散接触，将其运输穿过断裂约10次，然后在不等待可运输DNA片段耗尽的情况下从结合位点解离。
Type-II DNA topoisomerases resolve DNA entanglements such as supercoils, knots and catenanes by passing one segment of DNA duplex through a transient enzyme-bridged double-stranded break in another segment. The ATP-dependent passage reaction has previously been demonstrated at the single-molecule level, showing apparent processivity at saturating ATP. Here we directly observed the strand passage by human topoisomerase IIα, after winding a pair of fluorescently stained DNA molecules with optical tweezers for 30 turns into an X-shaped braid. On average 0.51±0.33 µm (11±6 turns) of a braid was unlinked in a burst of reactions taking 8±4 s, the unlinked length being essentially independent of the enzyme concentration between 0.25–37 pM. The time elapsed before the start of processive unlinking decreased with the enzyme concentration, being ∼100 s at 3.7 pM. These results are consistent with a scenario where the enzyme binds to one DNA for a period of ∼10 s, waiting for multiple diffusional encounters with the other DNA to transport it across the break ∼10 times, and then dissociates from the binding site without waiting for the exhaustion of transportable DNA segments.
登录
查看更多内容
DOI：
10.1073/pnas.1631550100
发表时间：
2003-08-19
影响因子：
11.1
作者：
Charvin, G;Bensimon, D;Croquette, V
通讯作者：
Croquette, V
DOI：
10.1073/pnas.0510509103
发表时间：
2006-02-07
影响因子：
11.1
作者：
Koster, DA;Wiggins, CH;Dekker, NH
通讯作者：
Dekker, NH
影响因子：
2.9
作者：
Renodon-Cornière, A;Jensen, LH;Sehested, M
通讯作者：
Sehested, M
影响因子：
4.8
作者：
McClendon, AK;Rodriguez, AC;Osheroff, N
通讯作者：
Osheroff, N
影响因子：
64.8
作者：
Yasuda, R;Noji, H;Itoh, H
通讯作者：
Itoh, H