Molecular mechanism of G-quadruplex unwinding helicase: sequential and repetitive unfolding of G-quadruplex by Pif1 helicase
Molecular mechanism of G-quadruplex unwinding helicase: sequential and repetitive unfolding of G-quadruplex by Pif1 helicase
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G-四链体解旋解旋酶的分子机制：Pif1解旋酶对G-四链体的顺序和重复解旋
DOI：
10.1042/bj20140997
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发表时间：
2015-02-15
影响因子：
4.1
通讯作者：
Xi, Xu-Guang
中科院分区：
文献类型：
--
作者：
Hou, Xi-Miao;Wu, Wen-Qiang;Xi, Xu-Guang
G-四链体 (G4) 研究的最新进展已证实 G4 结构存在于活细胞中，并可能对各种 DNA 事务产生有害影响。然而，解旋酶如何解析 G4 的研究才刚刚开始，并且在很大程度上仍然未知。在本文中，我们使用单分子荧光分析来探测 DNA 构建体中 Pif1 催化的 G4 解折叠，该 DNA 构建体类似于被 G4 停滞的滞后链的持续合成。引人注目的是，Pif1 展开，然后在 ss/dsDNA 连接处停止，随后 G4 快速重组，并通过同一单体“杂技”地重新启动展开。因此，Pif1 重复展开单个 G4 结构。此外，发现Pif1以两个大步骤顺序展开G4。我们的研究表明，作为一种稳定的中间体，G-三链体（G3）在此过程中发挥着重要作用。重复的展开活动可能有助于 Pif1 破坏不断重组的阻塞性 G4 结构，以挽救停滞的复制叉。所提出的 G4 逐步解折叠机制可能适用于解析 G4 结构以维持基因组稳定性的其他解旋酶。
Recent advances in G-quadruplex (G4) studies have confirmed that G4 structures exist in living cells and may have detrimental effects on various DNA transactions. How helicases resolve G4, however, has just begun to be studied and remains largely unknown. In the present paper, we use single-molecule fluorescence assays to probe Pif1-catalysed unfolding of G4 in a DNA construct resembling an ongoing synthesis of lagging strand stalled by G4. Strikingly, Pif1 unfolds and then halts at the ss/dsDNA junction, followed by rapid reformation of G4 and 'acrobatic' re-initiation of unfolding by the same monomer. Thus, Pif1 unfolds single G4 structures repetitively. Furthermore, it is found that Pif1 unfolds G4 sequentially in two large steps. Our study has revealed that, as a stable intermediate, G-triplex (G3) plays an essential role in this process. The repetitive unfolding activity may facilitate Pif1 disrupting the continuously reforming obstructive G4 structures to rescue a stalled replication fork. The proposed mechanism for step-wise unfolding of G4 is probably applicable to other helicases that resolve G4 structures for maintaining genome stability.