Single-Molecule Imaging of the Oligomer Formation of the Nonhexameric Escherichia coli UvrD Helicase
Single-Molecule Imaging of the Oligomer Formation of the Nonhexameric Escherichia coli UvrD Helicase
复制标题
DOI：
10.1016/j.bpj.2013.01.014
复制
发表时间：
2013-02-19
影响因子：
3.4
通讯作者：
Harada, Yoshie
中科院分区：
文献类型：
--
作者：
Yokota, Hiroaki;Chujo, Yuko Ayabe;Harada, Yoshie
超家族I解旋酶是负责核酸解绕的非六聚解旋酶。然而，它们是否以单体或低聚体的形式展开DNA仍然存在争议。在这项研究中，我们使用大肠杆菌UvrD（一种超家族I DNA解旋酶）的直接单分子荧光可视化来解决这个问题。我们对染料标记的解旋酶进行了光漂白步骤分析，并确定在没有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）双链DNA （dsDNA）和3'单链DNA （ssDNA）尾部（12,20或40 nt）结合。我们还发现，通过同时可视化解旋酶与DNA的结合/解离，以及在ATP存在下解旋酶的DNA解旋动力学，这些二聚体和三聚体形式负责DNA的解旋。因此，我们可以提出一种新的解旋酶-DNA相互作用的动力学方案，其中二聚体解旋酶和三聚体解旋酶都可以解旋DNA。据我们所知，这是第一个直接的单分子非六聚解旋酶定量研究，它有力地支持了一个模型，其中寡聚物是解旋酶的活性形式，这对所有超家族I解旋酶的DNA解绕机制具有重要意义。
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.