Belon Craig A, Frick David N
Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595, USA.
J Mol Biol. 2009 May 15;388(4):851-64. doi: 10.1016/j.jmb.2009.03.059. Epub 2009 Mar 28.
The hepatitis C virus (HCV) NS3 protein is a helicase capable of unwinding duplex RNA or DNA. This study uses a newly developed molecular-beacon-based helicase assay (MBHA) to investigate how nucleoside triphosphates (NTPs) fuel HCV helicase-catalyzed DNA unwinding. The MBHA monitors the irreversible helicase-catalyzed displacement of an oligonucleotide-bound molecular beacon so that rates of helicase translocation can be directly measured in real time. The MBHA reveals that HCV helicase unwinds DNA at different rates depending on the nature and concentration of NTPs in solution, such that the fastest reactions are observed in the presence of CTP followed by ATP, UTP, and GTP. 3'-Deoxy-NTPs generally support faster DNA unwinding, with dTTP supporting faster rates than any other canonical (d)NTP. The presence of an intact NS3 protease domain makes HCV helicase somewhat less specific than truncated NS3 bearing only its helicase region (NS3h). Various NTPs bind NS3h with similar affinities, but each NTP supports a different unwinding rate and processivity. Studies with NTP analogs reveal that specificity is determined by the nature of the Watson-Crick base-pairing region of the NTP base and the nature of the functional groups attached to the 2' and 3' carbons of the NTP sugar. The divalent metal bridging the NTP to NS3h also influences observed unwinding rates, with Mn(2+) supporting about 10 times faster unwinding than Mg(2+). Unlike Mg(2+), Mn(2+) does not support HCV helicase-catalyzed ATP hydrolysis in the absence of stimulating nucleic acids. Results are discussed in relation to models for how ATP might fuel the unwinding reaction.
丙型肝炎病毒（HCV）NS3蛋白是一种能够解开双链RNA或DNA的解旋酶。本研究使用一种新开发的基于分子信标的解旋酶检测方法（MBHA）来研究三磷酸核苷（NTP）如何为HCV解旋酶催化的DNA解旋提供能量。MBHA监测解旋酶催化的寡核苷酸结合分子信标的不可逆位移，从而可以实时直接测量解旋酶移位的速率。MBHA显示，HCV解旋酶以不同的速率解开DNA，这取决于溶液中NTP的性质和浓度，因此在CTP存在的情况下观察到最快的反应，其次是ATP、UTP和GTP。3'-脱氧-NTP通常支持更快的DNA解旋，dTTP支持的速率比任何其他标准（d）NTP都快。完整的NS3蛋白酶结构域的存在使HCV解旋酶的特异性略低于仅具有其解旋酶区域的截短型NS3（NS3h）。各种NTP以相似的亲和力结合NS3h，但每种NTP支持不同的解旋速率和持续合成能力。对NTP类似物的研究表明，特异性由NTP碱基的沃森-克里克碱基配对区域的性质以及连接到NTP糖的2'和3'碳上的官能团的性质决定。连接NTP与NS3h的二价金属也会影响观察到的解旋速率，Mn(2+)支持的解旋速率比Mg(2+)快约10倍。与Mg(2+)不同，在没有刺激核酸的情况下，Mn(2+)不支持HCV解旋酶催化的ATP水解。结合ATP如何为解旋反应提供能量的模型对结果进行了讨论。
