Laboratoire de physique de L'École Normale Supérieure de Paris, CNRS, ENS, Université PSL, Sorbonne Université, Université Paris Cité, 75005 Paris, France.
Institut de Biologie de l'École Normale Supérieure de Paris (IBENS), École Normale Supérieure, CNRS, INSERM, Université PSL, 75005 Paris, France.
Nucleic Acids Res. 2022 Aug 26;50(15):8767-8778. doi: 10.1093/nar/gkac667.
G-rich sequences found at multiple sites throughout all genomes may form secondary structures called G-quadruplexes (G4), which act as roadblocks for molecular motors. Among the enzymes thought to process these structures, the Pif1 DNA helicase is considered as an archetypical G4-resolvase and its absence has been linked to G4-related genomic instabilities in yeast. Here we developed a single-molecule assay to observe Pif1 opening a DNA duplex and resolving the G4 in real time. In support of former enzymological studies, we show that the helicase reduces the lifetime of G4 from hours to seconds. However, we observe that in the presence of a G4, Pif1 exhibits a strong strand switching behavior, which can lead to Pif1 escaping G4 resolution, depending on the structural context surrounding the substrate. This behavior is also detected in the presence of other roadblocks (LNA or RNA). We propose that the efficiency of Pif1 to remove a roadblock (G4 or other) is affected by its strand switching behavior and depends on the context surrounding the obstacle. We discuss how this switching behavior may explain several aspects of Pif1 substrate preference and affect its activity as a G4 resolvase in vivo.
富含 G 序列的结构在所有基因组的多个位点被发现，可能形成被称为 G-四链体（G4）的二级结构，其充当分子马达的障碍。在被认为处理这些结构的酶中，Pif1 DNA 解旋酶被认为是典型的 G4 解旋酶，其缺失与酵母中与 G4 相关的基因组不稳定性有关。在这里，我们开发了一种单分子测定法来实时观察 Pif1 打开 DNA 双链体并解析 G4。支持以前的酶学研究，我们表明该解旋酶将 G4 的寿命从数小时缩短至数秒。然而，我们观察到在 G4 的存在下，Pif1 表现出强烈的链交换行为，这可能导致 Pif1 逃避 G4 解析，具体取决于底物周围的结构上下文。在存在其他障碍（LNA 或 RNA）的情况下也检测到这种行为。我们提出，Pif1 去除障碍（G4 或其他）的效率受到其链交换行为的影响，并取决于障碍物周围的上下文。我们讨论了这种交换行为如何解释 Pif1 底物偏好的几个方面，并影响其作为体内 G4 解旋酶的活性。
