Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase.
Dda helicase tightly couples translocation on single-stranded DNA to unwinding of duplex DNA: Dda is an optimally active helicase.
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DOI：
10.1016/j.jmb.2012.04.007
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发表时间：
2012-07-13
影响因子：
5.6
通讯作者：
Raney, Kevin D.
中科院分区：
文献类型：
--
作者：
Byrd, Alicia K.;Matlock, Dennis L.;Bagchi, Debjani;Aarattuthodiyil, Suja;Harrison, David;Croquette, Vincent;Raney, Kevin D.
解旋酶利用ATP水解的能量解旋双链DNA，同时在DNA上移位。熔化双链体的机制已经被表征为主动或被动，这取决于酶是否主动分离碱基对或简单地隔离由于热磨损而形成的单链DNA。在这里，我们表明，Dda易位单向单链DNA在同一速率，它解开双链DNA在合奏和单分子实验。此外，解绕速率在很大程度上对双链体稳定性和所施加的力不敏感。因此，Dda将其所有易位酶活性转换成DNA解旋活性，使得解旋速率受到易位速率的限制，并且酶主动分离双链体。主动解旋酶和被动解旋酶的特征在于，DNA解旋速度（单位为每秒碱基对）（Vun）除以单链DNA上的易位速度（单位为每秒核苷酸）（Vtranss）。如果所得分数为0.25，则认为解旋酶处于“活性”范围的下端。在Dda的情况下，平均DNA解旋速度为257 ± 42 bp/s，平均易位速度为267 ± 15个核苷酸/s。0.96的Vun/Vtranss值将Dda置于作为基本上“完美”活性解旋酶的独特类别中。
Helicases utilize the energy of ATP hydrolysis to unwind double-stranded DNA while translocating on the DNA. Mechanisms for melting the duplex have been characterized as active or passive, depending on whether the enzyme actively separates the base pairs or simply sequesters single-stranded DNA that forms due to thermal fraying. Here we show that Dda translocates unidirectionally on single-stranded DNA at the same rate at which it unwinds double-stranded DNA in both ensemble and single molecule experiments. Further, the unwinding rate is largely insensitive to the duplex stability and to applied force. Thus, Dda transduces all of its translocase activity into DNA unwinding activity so that the rate of unwinding is limited by the rate of translocation and the enzyme actively separates the duplex. Active and passive helicases have been characterized by dividing the velocity of DNA unwinding in base pairs per second (Vun) by the velocity of translocation on single-stranded DNA in nucleotides per second (Vtrans). If the resulting fraction is 0.25, then a helicase is considered to be at the lower end of the “active” range. In the case of Dda, the average DNA unwinding velocity was 257 ± 42 bp/s and the average translocation velocity was 267 ± 15 nucleotides/s. The Vun/Vtrans value of 0.96 places Dda in a unique category of being an essentially “perfectly” active helicase.
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