Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Kyoto, Japan.
Biophys J. 2013 Feb 19;104(4):924-33. doi: 10.1016/j.bpj.2013.01.014.
Superfamily I helicases are nonhexameric helicases responsible for the unwinding of nucleic acids. However, whether they unwind DNA in the form of monomers or oligomers remains a controversy. In this study, we addressed this question using direct single-molecule fluorescence visualization of Escherichia coli UvrD, a superfamily I DNA helicase. We performed a photobleaching-step analysis of dye-labeled helicases and determined that the helicase is bound to 18-basepair (bp) double-stranded DNA (dsDNA) with a 3' single-stranded DNA (ssDNA) tail (12, 20, or 40 nt) in a dimeric or trimeric form in the absence of ATP. We also discovered through simultaneous visualization of association/dissociation of the helicase with/from DNA and the DNA unwinding dynamics of the helicase in the presence of ATP that these dimeric and trimeric forms are responsible for the unwinding of DNA. We can therefore propose a new kinetic scheme for the helicase-DNA interaction in which not only a dimeric helicase but also a trimeric helicase can unwind DNA. This is, to our knowledge, the first direct single-molecule nonhexameric helicase quantification study, and it strongly supports a model in which an oligomer is the active form of the helicase, which carries important implications for the DNA unwinding mechanism of all superfamily I helicases.
超家族 I 解旋酶是非六聚体解旋酶，负责解开核酸。然而，它们是以单体还是寡聚体的形式解开 DNA 仍然存在争议。在这项研究中，我们使用直接的单分子荧光可视化方法研究了大肠杆菌 UvrD，一种超家族 I DNA 解旋酶，解决了这个问题。我们对染料标记的解旋酶进行了光漂白步分析，确定在没有 ATP 的情况下，解旋酶以二聚体或三聚体的形式与 18 碱基对 (bp) 的双链 DNA (dsDNA)结合，带有 3'单链 DNA (ssDNA) 尾 (12、20 或 40 个核苷酸)。我们还通过同时可视化解旋酶与 DNA 的结合/解离以及解旋酶在 ATP 存在下的 DNA 解旋动力学，发现这些二聚体和三聚体形式负责 DNA 的解旋。因此，我们可以提出一个新的解旋酶-DNA 相互作用的动力学方案，其中不仅二聚体解旋酶，而且三聚体解旋酶也可以解开 DNA。据我们所知，这是第一个直接的单分子非六聚体解旋酶定量研究，它强烈支持寡聚体是解旋酶的活性形式的模型，这对所有超家族 I 解旋酶的 DNA 解旋机制具有重要意义。
