Porter D J, Preugschat F
Glaxo Wellcome, 5 Moore Drive, Research Triangle Park, North Carolina 27709, USA.
Biochemistry. 2000 May 2;39(17):5166-73. doi: 10.1021/bi992384f.
HCV helicase [E(wt)] catalyzed strand separation of a short DNA duplex (F21:HF31) formed from a 5'-hexachlorofluorescein-tagged 31-mer (HF31) and a 3'-fluorescein-tagged 21-mer (F21) complementary to the 5'-end of HF31. Strand separation was monitored by the fluorescence increase associated with the formation of F21 from F21:HF31. In the presence of ATP, the strand-separating activity was catalytic. In the absence of ATP and with E(wt) concentrations greater than that of F21:HF31, a biphasic fluorescence increase was observed at 25 degrees C. The late phase of this reaction was assigned to the separation of F21 from F21:HF31. The ATP-independent strand-separating reaction occurred more rapidly in the absence of Mg(2+) than in its presence. This result correlated with a lower T(m) value of F21:HF31 in the absence of 3.5 mM Mg(2+) than in its presence (45 vs 63 degrees C). The stoichiometry for the strand-separating reaction in the absence of ATP was 8 mol of E(wt) per mole of F21:HF31 separated into single-stranded F21 and HF31. The dissociation constants of HCV helicase for F21, HF31, and F21:HF31 in the absence of Mg(2+) were 0.6 +/- 0.4, 6 +/- 1, and 7.3 +/- 0.9 nM, respectively. Histidinyl-tagged E(wt) [hE(wt)] and a mutant enzyme [hE(V432A)] were prepared. hE(wt) and E(wt) bound F21 and HF31 with similar affinities and had similar ATP-dependent helicase activities, whereas hE(V432A) bound F21 and HF31 with affinities similar to that of E(wt) but had greatly reduced ATP-dependent helicase activities. In contrast to E(wt) and hE(wt), hE(V432A) did not support the ATP-independent strand-separating reaction. Consequently, the ATP-independent strand-separating reaction was not only the result of the high affinity of the enzyme for single-stranded DNA. The enzyme preferentially used duplex DNA with a 3'-tail for the ATP-dependent helicase reaction. In contrast, the enzyme strand-separated blunt-ended, 5'-tailed, and 3'-tailed duplex DNA equally effectively in the ATP-independent strand-separating reaction.
丙型肝炎病毒解旋酶[E(wt)]催化由一个5'-六氯荧光素标记的31聚体(HF31)和一个与HF31的5'-末端互补的3'-荧光素标记的21聚体(F21)形成的短DNA双链体(F21:HF31)的链分离。通过与F21:HF31形成F21相关的荧光增加来监测链分离。在ATP存在下，链分离活性是催化性的。在没有ATP且E(wt)浓度大于F21:HF31浓度的情况下，在25℃观察到双相荧光增加。该反应的后期被归因于F21与F21:HF31的分离。与存在Mg(2+)时相比，在没有Mg(2+)的情况下，不依赖ATP的链分离反应发生得更快。该结果与在没有3.5 mM Mg(2+)时F21:HF31的较低解链温度值相关，而存在Mg(2+)时为45℃，不存在时为63℃。在没有ATP的情况下，链分离反应的化学计量比为每摩尔分离成单链F21和HF31的F21:HF31有8摩尔E(wt)。在没有Mg(2+)的情况下，丙型肝炎病毒解旋酶对F21、HF31和F21:HF31的解离常数分别为0.6±0.4、6±1和7.3±0.9 nM。制备了组氨酸标签化的E(wt)[hE(wt)]和一种突变酶[hE(V432A)]。hE(wt)和E(wt)以相似的亲和力结合F21和HF31，并且具有相似的ATP依赖性解旋酶活性，而hE(V432A)以与E(wt)相似的亲和力结合F21和HF31，但具有大大降低的ATP依赖性解旋酶活性。与E(wt)和hE(wt)相反，hE(V432A)不支持不依赖ATP的链分离反应。因此，不依赖ATP的链分离反应不仅仅是酶对单链DNA高亲和力的结果。该酶在ATP依赖性解旋酶反应中优先使用具有3'-尾的双链DNA。相反，在不依赖ATP的链分离反应中，该酶对平端、5'-尾和3'-尾双链DNA的链分离效果相同。
