Jennings Thomas A, Mackintosh Samuel G, Harrison Melody K, Sikora Deniz, Sikora Bartek, Dave Bhuvanesh, Tackett Alan J, Cameron Craig E, Raney Kevin D
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
J Biol Chem. 2009 Feb 20;284(8):4806-14. doi: 10.1074/jbc.M805540200. Epub 2008 Dec 16.
Hepatitis C virus NS3 helicase can unwind double-stranded DNA and RNA and has been proposed to form oligomeric structures. Here we examine the DNA unwinding activity of monomeric NS3. Oligomerization was measured by preparing a fluorescently labeled form of NS3, which was titrated with unlabeled NS3, resulting in a hyperbolic increase in fluorescence anisotropy and providing an apparent equilibrium dissociation constant of 236 nm. To evaluate the DNA binding activity of individual subunits within NS3 oligomers, two oligonucleotides were labeled with fluorescent donor or acceptor molecules and then titrated with NS3. Upon the addition of increasing concentrations of NS3, fluorescence energy transfer was observed, which reached a plateau at a 1:1 ratio of NS3 to oligonucleotides, indicating that each subunit within the oligomeric form of NS3 binds to DNA. DNA unwinding was measured under multiple turnover conditions with increasing concentrations of NS3; however, no increase in specific activity was observed, even at enzyme concentrations greater than the apparent dissociation constant for oligomerization. An ATPase-deficient form of NS3, NS3(D290A), was prepared to explore the functional consequences of oligomerization. Under single turnover conditions in the presence of excess concentration of NS3 relative to DNA, NS3(D290A) exhibited a dominant negative effect. However, under multiple turnover conditions in which DNA concentration was in excess to enzyme concentration, NS3(D290A) did not exhibit a dominant negative effect. Taken together, these data support a model in which monomeric forms of NS3 are active. Oligomerization of NS3 occurs, but subunits can function independently or cooperatively, dependent upon the relative concentration of the DNA.
丙型肝炎病毒NS3解旋酶能够解开双链DNA和RNA，并被认为会形成寡聚体结构。在此，我们研究了单体NS3的DNA解旋活性。通过制备一种荧光标记形式的NS3来测量寡聚化，用未标记的NS3对其进行滴定，导致荧光偏振呈双曲线增加，并得出表观平衡解离常数为236纳米。为了评估NS3寡聚体内各个亚基的DNA结合活性，用荧光供体或受体分子标记两条寡核苷酸，然后用NS3进行滴定。随着NS3浓度的增加，观察到荧光能量转移，当NS3与寡核苷酸的比例为1:1时达到平台期，表明NS3寡聚体形式中的每个亚基都与DNA结合。在多种周转条件下，随着NS3浓度的增加测量DNA解旋；然而，即使在酶浓度大于寡聚化的表观解离常数时，也未观察到比活性增加。制备了一种ATP酶缺陷型的NS3，即NS3(D290A)，以探究寡聚化的功能后果。在相对于DNA存在过量浓度NS3的单周转条件下，NS3(D290A)表现出显性负效应。然而，在DNA浓度超过酶浓度的多种周转条件下，NS3(D290A)未表现出显性负效应。综上所述，这些数据支持一个模型，即NS3的单体形式具有活性。NS3会发生寡聚化，但亚基可以独立或协同发挥作用，这取决于DNA的相对浓度。
