Matson S W, George J W
J Biol Chem. 1987 Feb 15;262(5):2066-76.
Escherichia coli helicase II has been purified to near homogeneity from cells harboring a multicopy plasmid containing the structural gene for helicase II, uvrD. In this paper a detailed description of the single-stranded DNA-dependent nucleoside 5'-triphosphatase and helicase reactions catalyzed by helicase II is presented. The results of this study suggest that nucleoside 5'-triphosphate hydrolysis provides the energy required for translocation of the enzyme along single-stranded DNA. Measurements of the rate of ATP hydrolysis using a variety of single-stranded DNAs of known structure and length suggest a processive translocation mechanism for helicase II. Single-stranded DNA coated with either Escherichia coli single-stranded DNA binding protein (SSB) or bacteriophage T4 gene 32 protein fails to support helicase II ATPase activity. Moreover, helicase II is apparently unable to displace a molecule of bound SSB protein from single-stranded DNA when it is encountered in the process of translocation along a single-stranded DNA effector. The helicase reaction has been characterized using an in vitro strand displacement helicase assay. The helicase reaction requires concomitant nucleoside 5'-triphosphatase hydrolysis that is satisfied by the hydrolysis of either rATP or dATP. As the length of duplex DNA present in the partial duplex helicase substrate is increased from 71 base pairs to 343 base pairs, the fraction of duplex DNA molecules that are unwound by helicase II decreases in the absence of any accessory proteins. However, the total number of base pairs of duplex DNA unwound depends primarily on the amount of enzyme added to the helicase reaction and not on the length of the duplex DNA present in the partial duplex DNA substrate. These data suggest the number of base pairs of duplex DNA unwound is directly proportional with the concentration of helicase II in the reaction mixture. In addition, the rate of the unwinding reaction is independent of the length of the duplex DNA available for unwinding. Helicase II has been shown to dissociate from single-stranded DNA molecules infrequently acting as an ATPase. However, the enzyme dissociates from partial duplex helicase substrates more frequently. This suggests a more distributive reaction mechanism on duplex DNA than was observed on single-stranded DNA substrates. The fraction of 343-base pair partial duplex DNA molecules unwound by helicase II can be increased by the addition of appropriate concentrations of E. coli SSB to the reaction. This suggests that helicase II and SSB may act in a concerted reaction to unwind duplex DNA.
已从携带含有解旋酶II（uvrD）结构基因的多拷贝质粒的细胞中，将大肠杆菌解旋酶II纯化至近乎均一的状态。本文详细描述了解旋酶II催化的单链DNA依赖性核苷5'-三磷酸酶和螺旋酶反应。这项研究的结果表明，核苷5'-三磷酸水解为该酶沿单链DNA的移位提供了所需能量。使用各种已知结构和长度的单链DNA对ATP水解速率进行的测量表明，解旋酶II存在一种持续性移位机制。被大肠杆菌单链DNA结合蛋白（SSB）或噬菌体T4基因32蛋白包被的单链DNA无法支持解旋酶II的ATP酶活性。此外，当解旋酶II在沿单链DNA效应物移位过程中遇到结合在单链DNA上的SSB蛋白分子时，它显然无法将其取代。已使用体外链置换螺旋酶测定法对螺旋酶反应进行了表征。螺旋酶反应需要伴随核苷5'-三磷酸酶水解，rATP或dATP的水解均可满足这一需求。随着部分双链螺旋酶底物中双链DNA的长度从71个碱基对增加到343个碱基对，在没有任何辅助蛋白的情况下，被解旋酶II解开的双链DNA分子比例会降低。然而，解开的双链DNA碱基对总数主要取决于添加到螺旋酶反应中的酶量，而不取决于部分双链DNA底物中双链DNA的长度。这些数据表明，解开的双链DNA碱基对数量与反应混合物中解旋酶II的浓度成正比。此外，解旋反应的速率与可供解开的双链DNA长度无关。已证明解旋酶II很少从单链DNA分子上解离并作为ATP酶起作用。然而，该酶从部分双链螺旋酶底物上解离的频率更高。这表明与在单链DNA底物上观察到的情况相比，在双链DNA上存在一种更分散的反应机制。通过向反应中添加适当浓度的大肠杆菌SSB，可以增加被解旋酶II解开的343个碱基对部分双链DNA分子的比例。这表明解旋酶II和SSB可能协同作用以解开双链DNA。
