Continuous assays for DNA translocation using fluorescent triplex dissociation: Application to type I restriction endonucleases
Continuous assays for DNA translocation using fluorescent triplex dissociation: Application to type I restriction endonucleases
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DOI：
10.1016/j.jmb.2005.03.018
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发表时间：
2005-05-13
影响因子：
5.6
通讯作者：
Szczelkun, MD
中科院分区：
文献类型：
--
作者：
McClelland, SE;Dryden, DTF;Szczelkun, MD
描述了独立于双链解离的DNA易位分析的荧光分析和伴随的动力学模型。在IA、IB和IC型限制性内切酶(分别为EcoKI、EcoAI和EcoR1241)识别序列下游的DNA底物上引入了一个三链结合位点(TBS)。每个内切酶都与底物孵育(不含ATP)，底物上预先结合了六氯荧光素标记的三链形成寡核苷酸(HEX-TFO)。在加入三磷酸腺苷后，一维的酶运动导致与HEX-TFO的碰撞和置换，导致荧光强度增加两倍。或者，监测了罗丹明标记的TFO置换后各向异性的降低。利用停流荧光计中的快速混合，产生了连续的动力学分布，其中置换之前有一个滞后阶段，与移动的距离成正比。对于每种酶，我们不仅获得了易位。此外，还包括关于启动时缓慢异构化步骤(S)的信息。此外，我们还证明了缺乏DNA切割但ATPase活性最高的酶的启动和转位速率与野生型相同，证实了DNA链断裂不是运动的先决条件。(C)2005爱思唯尔有限公司。保留所有权利。
Fluorescent assays and accompanying kinetic models are described for the analysis of DNA translocation independent of duplex unwinding. A triplex binding site (TBS) was introduced into DNA substrates at precise loci downstream of recognition sequences for type IA, IB and IC restriction endonucleases (EcoKI, EcoAI and EcoR1241, respectively). Each endonuclease was incubated (without ATP) with substrates on which a hexachlorofluoroscein-labelled triplex-forming oligonucleotide (HEX-TFO) was pre-bound. Following addition of ATP, 1-D enzyme motion resulted in collision with, and displacement of, the HEX-TFO, producing a > twofold increase in fluorescent intensity. Alternatively, a decrease in anisotropy following displacement of a rhodamine-labelled TFO was monitored. Using rapid mixing in a stopped-flow fluorimeter, continuous kinetic profiles were produced in which displacement is preceded by a lag-phase, directly proportional to the distance moved. For each enzyme, we obtained not only the translocation. rate but also information on slow isomerisation step(s) at initiation. Furthermore, we demonstrated that enzymes deficient in DNA cleavage but with maximal ATPase activity showed initiation and translocation rates identical to wild-type, confirming that DNA strand breaks are not a pre-requisite of motion. (c) 2005 Elsevier Ltd. All rights reserved.