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首页> 外文期刊>Biochemistry >Strand-separating activity of hepatitis C virus helicase in the absence of ATP.
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Strand-separating activity of hepatitis C virus helicase in the absence of ATP.

机译：在没有ATP的情况下丙型肝炎病毒解旋酶的链分离活性。

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摘要

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.
机译：HCV解旋酶[E（wt）]催化由5'-六氯荧光素标记的31-mer（HF31）和3'-荧光素标记的21-mer（F21）形成的短DNA双链体（F21：HF31）的链分离与HF31的5'端互补。通过与由F21：HF31形成F21相关的荧光增加来监测链分离。在ATP存在下，链分离活性是催化的。在不存在ATP且E（wt）浓度大于F21：HF31的情况下，在25°C时观察到双相荧光增加。该反应的后期是将F21与F21：HF31分离。不存在Mg（2+）的情况下，ATP独立的链分离反应发生的速度比存在它的速度更快。此结果与在不存在3.5 mM Mg（2+）的情况下F21：HF31的T（m）值比存在时更低（45 vs 63摄氏度）相关。在不存在ATP的情况下，链分离反应的化学计量为每摩尔分离为单链F21和HF31的F21：HF31每摩尔8摩尔E（wt）。在没有Mg（2+）的情况下，F21，HF31和F21：HF31的HCV解旋酶的解离常数分别为0.6 +/- 0.4、6 +/- 1和7.3 +/- 0.9 nM。制备了带有组氨酸标记的E（wt）[hE（wt）]和突变酶[hE（V432A）]。 hE（wt）和E（wt）以相似的亲和力结合F21和HF31，并具有相似的ATP依赖解旋酶活性，而hE（V432A）以相似于E（wt）的亲和力结合F21和HF31，但ATP-依赖性解旋酶活性。与E（wt）和hE（wt）相比，hE（V432A）不支持不依赖ATP的链分离反应。因此，不依赖ATP的链分离反应不仅是酶对单链DNA的高亲和力的结果。该酶优先使用具有3'尾巴的双链DNA进行ATP依赖的解旋酶反应。相反，在不依赖ATP的链分离反应中，酶链分离的平末端，5'尾和3'尾双链DNA同样有效。

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