Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.
Center for Science and Engineering of Living Systems, Washington University in St. Louis, St. Louis, MO 63130.
Proc Natl Acad Sci U S A. 2019 Dec 3;116(49):24533-24541. doi: 10.1073/pnas.1915654116. Epub 2019 Nov 19.
Members of the Pif1 family of helicases function in multiple pathways that involve DNA synthesis: DNA replication across G-quadruplexes; break-induced replication; and processing of long flaps during Okazaki fragment maturation. Furthermore, Pif1 increases strand-displacement DNA synthesis by DNA polymerase δ and allows DNA replication across arrays of proteins tightly bound to DNA. This is a surprising feat since DNA rewinding or annealing activities limit the amount of single-stranded DNA product that Pif1 can generate, leading to an apparently poorly processive helicase. In this work, using single-molecule Förster resonance energy transfer approaches, we show that 2 members of the Pif1 family of helicases, Pif1 from and Pfh1 from , unwind double-stranded DNA by a branched mechanism with 2 modes of activity. In the dominant mode, only short stretches of DNA can be processively and repetitively opened, with reclosure of the DNA occurring by mechanisms other than strand-switching. In the other less frequent mode, longer stretches of DNA are unwound via a path that is separate from the one leading to repetitive unwinding. Analysis of the kinetic partitioning between the 2 different modes suggests that the branching point in the mechanism is established by conformational selection, controlled by the interaction of the helicase with the 3' nontranslocating strand. The data suggest that the dominant and repetitive mode of DNA opening of the helicase can be used to allow efficient DNA replication, with DNA synthesis on the nontranslocating strand rectifying the DNA unwinding activity.
Pif1 家族的解旋酶成员在多个涉及 DNA 合成的途径中发挥作用：跨越 G-四联体的 DNA 复制；断裂诱导复制；以及在冈崎片段成熟过程中处理长瓣。此外，Pif1 通过 DNA 聚合酶 δ 增加链置换 DNA 合成，并允许 DNA 跨越紧密结合在 DNA 上的蛋白质阵列复制。这是一项令人惊讶的壮举，因为 DNA 重绕或退火活性限制了 Pif1 可以产生的单链 DNA 产物的量，导致其解旋酶的活性明显较差。在这项工作中，我们使用单分子Förster 共振能量转移方法表明，Pif1 家族的 2 个解旋酶成员，来自 和 Pfh1 ，通过具有 2 种活性模式的分支机制解开双链 DNA。在主导模式下，只有短片段的 DNA 可以进行重复和连续打开，DNA 的重新闭合通过除链交换以外的机制发生。在另一种不太常见的模式下，较长的 DNA 片段通过与导致重复解开的路径分开的路径解开。对 2 种不同模式之间的动力学分配的分析表明，机制中的分支点是由构象选择建立的，由解旋酶与 3'非转移链的相互作用控制。数据表明，解旋酶的主导和重复 DNA 打开模式可用于允许有效的 DNA 复制，非转移链上的 DNA 合成纠正 DNA 解开活性。
