Escherichia coli Rep protein and helicase IV. Distributive single-stranded DNA-dependent ATPases that catalyze a limited unwinding reaction in vitro.
Escherichia coli Rep protein and helicase IV. Distributive single-stranded DNA-dependent ATPases that catalyze a limited unwinding reaction in vitro.
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大肠杆菌 Rep 蛋白和解旋酶 IV。
DOI：
10.1111/j.1432-1033.1992.tb17074.x
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Matson,SW
中科院分区：
文献类型：
--
作者：
Yancey-Wrona,JE;Wood,ER;George,JW;Smith,KR;Matson,SW
Rep蛋白和解旋酶IV是两种具有解旋酶活性的DNA依赖性腺苷5′-三磷酸酶，已从大肠杆菌中纯化并鉴定。这两种酶都表现出与单链DNA的分布性相互作用，作为DNA依赖性ATP酶，在反应中对NaCl浓度的增加具有相对抗性，对E的加入敏感。大肠杆菌单链DNA结合蛋白（SSB）。每种蛋白质催化的解旋酶反应的特征在于使用直接解旋测定和部分双链体DNA底物。Rep蛋白和解旋酶IV都催化长度为71 bp的双链体区域的解旋。然而，与71-bp底物的解旋相比，119-bp或343-bp双链体区域的解旋显著减少。在检测的各蛋白浓度下，使用71-bp底物时解绕的碱基巴黎数最多，使用119-bp底物时居中，使用343-bp底物时最少。添加E. coliSSB没有增加Rep蛋白解绕的343个核苷酸片段的分数。然而，添加SSB确实刺激解旋酶IV催化的解旋反应约两倍。此外，稳定双链体DNA的离子强度条件（即加入MgCl 2或NaCl），显着抑制由Rep蛋白或解旋酶IV催化的解旋酶反应，而对ATP酶反应几乎没有影响。因此，这两种酶似乎共享用于解旋双链体DNA的共同生物化学机制，其可以被描述为双链体DNA的有限解旋。综上所述，这些数据表明，在体外，并在不存在额外的蛋白质，无论是Rep蛋白，也不解旋酶IV催化的进行性解旋反应。
Rep protein and helicase IV, two DNA‐dependent adenosine 5′‐triphosphatases with helicase activity, have been purified fromEscherichia coliand characterized. Both enzymes exhibit a distributive interaction with single‐stranded DNA as DNA‐dependent ATPases in a reaction that is relatively resistant to increasing NaCl concentration and sensitive to the addition ofE. Colisingle‐stranded DNA binding protein (SSB). The helicase reaction catalyzed by each protein has been characterized using a direct unwinding assay and partial duplex DNA substrates. Both Rep protein and helicase IV catalyzed the unwinding of a duplex region 71 bp in length. However, unwinding of a 119‐bp or 343‐bp duplex region was substantially reduced compared to unwinding of the 71‐bp substrate. At each concentration of protein examined, the number of base paris unwound was greatest using the 71‐bp substrate, intermediate with the 119‐bp substrate and lowest using the 343‐bp substrate. The addition ofE. coliSSB did not increase the fraction of the 343‐nucleotide fragment unwound by Rep protein. However, the addition of SSB did stimulate the unwinding reaction catalyzed by helicase IV approximately twofold. In addition, ionic strength conditions which stabilize duplex DNA (i.e. addition of MgCl2or NaCl), markedly inhibited the helicase reaction catalyzed by either Rep protein or helicase IV while having little effect on the ATPase reaction. Thus, these two enzymes appear to share a common biochemical mechanism for unwinding duplex DNA which can be described as limited unwinding of duplex DNA. Taken together these data suggest that,in vitro, and in the absence of addition proteins, neither Rep protein nor helicase IV catalyzes a processive unwinding reaction.