Department of Physics, University of Illinois, Urbana-Champaign, Urbana, United States.
Center for Biophysics and Quantitative Biology, University of Illinois, Urbana-Champaign, Urbana, United States.
Elife. 2021 Mar 19;10:e60515. doi: 10.7554/eLife.60515.
Helicases utilize nucleotide triphosphate (NTP) hydrolysis to translocate along single-stranded nucleic acids (NA) and unwind the duplex. In the cell, helicases function in the context of other NA-associated proteins such as single-stranded DNA binding proteins. Such encounters regulate helicase function, although the underlying mechanisms remain largely unknown. xeroderma pigmentosum group D (XPD) helicase serves as a model for understanding the molecular mechanisms of superfamily 2B helicases, and its activity is enhanced by the cognate single-stranded DNA binding protein replication protein A 2 (RPA2). Here, optical trap measurements of the unwinding activity of a single XPD helicase in the presence of RPA2 reveal a mechanism in which XPD interconverts between two states with different processivities and transient RPA2 interactions stabilize the more processive state, activating a latent 'processivity switch' in XPD. A point mutation at a regulatory DNA binding site on XPD similarly activates this switch. These findings provide new insights on mechanisms of helicase regulation by accessory proteins.
解旋酶利用核苷酸三磷酸（NTP）水解沿单链核酸（NA）移动并解双链。在细胞中，解旋酶在其他与 NA 相关的蛋白质（如单链 DNA 结合蛋白）的背景下发挥作用。这些相互作用调节解旋酶的功能，尽管其潜在机制在很大程度上仍不清楚。着色性干皮病组 D（XPD）解旋酶可作为理解超家族 2B 解旋酶分子机制的模型，其活性通过同源单链 DNA 结合蛋白复制蛋白 A2（RPA2）增强。在这里，在存在 RPA2 的情况下，对单个 XPD 解旋酶的解旋活性的光学捕获测量揭示了一种机制，其中 XPD 在两种具有不同延伸性的状态之间转换，并且瞬态 RPA2 相互作用稳定了更具延伸性的状态，激活了 XPD 中的潜在“延伸性开关”。在 XPD 上的调节 DNA 结合位点的点突变同样激活了此开关。这些发现为辅助蛋白调节解旋酶的机制提供了新的见解。
