PROPERTIES OF THE DUPLEX DNA-DEPENDENT ATPASE ACTIVITY OF ESCHERICHIA-COLI RECA PROTEIN AND ITS ROLE IN BRANCH MIGRATION
PROPERTIES OF THE DUPLEX DNA-DEPENDENT ATPASE ACTIVITY OF ESCHERICHIA-COLI RECA PROTEIN AND ITS ROLE IN BRANCH MIGRATION
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DOI：
10.1073/pnas.84.10.3127
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发表时间：
1987-05-01
影响因子：
11.1
通讯作者：
KRUPP, RA
中科院分区：
文献类型：
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作者：
KOWALCZYKOWSKI, SC;CLOW, J;KRUPP, RA
我们研究了recA蛋白的双链DNA（dsDNA）依赖的ATP酶活性。这种活性与单链DNA（ssDNA）依赖性ATP酶活性的区别在于，在稳态ATP水解开始之前存在明显的滞后时间。在滞后期，几乎没有ATP水解。滞后期的持续时间，被称为滞后时间，被发现随着dsDNA底物的热稳定性而增加。增加MgCl 2或NaCl浓度增加滞后时间，而增加温度减少滞后时间。滞后时间对recA蛋白浓度的依赖性很小，但强烈依赖于ATP浓度。在滞后期之后，达到接近用ssDNA观察到的速率的稳态ATP水解速率。反应的稳态阶段与recA蛋白-DNA复合物的浓度成比例，并在约20 ℃时显示饱和行为。5 .+-。每个recA蛋白单体1个碱基对。这些结果表明，滞后期代表了dsDNA依赖性ATP水解反应中的限速步骤，该反应需要dsDNA中的结构转变，并且涉及ATP、recA蛋白和DNA的三元复合物。我们认为这种转变涉及dsDNA的瞬时变性，以形成ssDNA的区域。在其他地方，我们证明了dsDNA依赖的ATP酶活性与recA蛋白催化的分支迁移率成正比。我们认为，这种活动是负责极性聚合，驱动的分支迁移反应。
We have investigated the double-stranded DNA (dsDNA)-dependent ATPase activity of recA protein. This activity is distinguished from the single-stranded DNA (ssDNA)-dependent ATPase activity by the presence of a pronounced lag time before the onset of steady-state ATP hydrolysis. During the lag phase there is little ATP hydrolysis. The duration of the lag phase, referred to as the lag time, is found to increase with the thermal stability of the dsDNA substrate. Increasing either the MgCl2 or NaCl concentration increases the lag time, whereas increasing the temperature decreases the lag time. The lag time shows little dependence on recA protein concentration but is strongly dependent on ATP concentration. After the lag phase, steady-state ATP hydrolysis rate is achieved that approaches the rate observed with ssDNA. The steady-state phase of the reaction is proportional to the concentration of recA protein-DNA complex and shows saturation behavior at .apprxeq. 5 .+-. 1 base pairs per recA protein monomer. These results suggest that the lag phase represents a rate-limiting step in the dsDNA-dependent ATP hydrolysis reaction that requires a structural transition in the dsDNA and that involves a ternary complex of ATP, recA protein, and DNA. We propose that this transition involves the transient denaturation of the dsDNA to form regions of ssDNA. Elsewhere we demonstrate that the dsDNA-dependent ATPase activity is proportional to the rate of recA protein-catalyzed branch migration. We suggest that this activity is responsible for a polar polymerization that drives the branch migration reaction.