Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, Saint Louis, MO 63110, USA.
Department of Biochemistry & Molecular Biophysics, Washington University School of Medicine, 660 S. Euclid Ave, Saint Louis, MO 63110, USA; Biochemistry & Biophysics, Blueprint Medicines, Cambridge, MA 02139, USA.
J Mol Biol. 2024 Jun 1;436(11):168578. doi: 10.1016/j.jmb.2024.168578. Epub 2024 Apr 20.
Monomers of the Superfamily (SF) 1 helicases, E. coli Rep and UvrD, can translocate directionally along single stranded (ss) DNA, but must be activated to function as helicases. In the absence of accessory factors, helicase activity requires Rep and UvrD homo-dimerization. The ssDNA binding sites of SF1 helicases contain a conserved aromatic amino acid (Trp250 in Rep and Trp256 in UvrD) that stacks with the DNA bases. Here we show that mutation of this Trp to Ala eliminates helicase activity in both Rep and UvrD. Rep(W250A) and UvrD(W256A) can still dimerize, bind DNA, and monomers still retain ATP-dependent ssDNA translocase activity, although with ∼10-fold lower rates and lower processivities than wild type monomers. Although neither wtRep monomers nor Rep(W250A) monomers possess helicase activity by themselves, using both ensemble and single molecule methods, we show that helicase activity is achieved upon formation of a Rep(W250A)/wtRep hetero-dimer. An ATPase deficient Rep monomer is unable to activate a wtRep monomer indicating that ATPase activity is needed in both subunits of the Rep hetero-dimer. We find the same results with E. coli UvrD and its equivalent mutant (UvrD(W256A)). Importantly, Rep(W250A) is unable to activate a wtUvrD monomer and UvrD(W256A) is unable to activate a wtRep monomer indicating that specific dimer interactions are required for helicase activity. We also demonstrate subunit communication within the dimer by virtue of Trp fluorescence signals that only are present within the Rep dimer, but not the monomers. These results bear on proposed subunit switching mechanisms for dimeric helicase activity.
SF1 家族单体解旋酶，大肠杆菌 Rep 和 UvrD，可以沿单链 DNA 定向迁移，但必须被激活才能发挥解旋酶的功能。在没有辅助因子的情况下，解旋酶活性需要 Rep 和 UvrD 同源二聚化。SF1 解旋酶的 ssDNA 结合位点包含一个保守的芳香族氨基酸（Rep 中的色氨酸 250 和 UvrD 中的色氨酸 256），与 DNA 碱基堆叠。在这里，我们表明，将该色氨酸突变为丙氨酸会消除 Rep 和 UvrD 中的解旋酶活性。Rep（W250A）和 UvrD（W256A）仍然可以二聚化，结合 DNA，并且单体仍然保留 ATP 依赖性 ssDNA 易位酶活性，尽管比野生型单体的速率和进程低约 10 倍。尽管 wtRep 单体或 Rep（W250A）单体本身都没有解旋酶活性，但我们使用集总和单分子方法表明，在形成 Rep（W250A）/wtRep 异源二聚体时会产生解旋酶活性。缺乏 ATPase 的 Rep 单体无法激活 wtRep 单体，表明 Rep 异源二聚体的两个亚基都需要 ATPase 活性。我们在大肠杆菌 UvrD 及其等效突变体（UvrD（W256A））中发现了相同的结果。重要的是，Rep（W250A）无法激活 wtUvrD 单体，UvrD（W256A）无法激活 wtRep 单体，表明需要特定的二聚体相互作用才能产生解旋酶活性。我们还通过仅存在于 Rep 二聚体而不存在于单体中的色氨酸荧光信号证明了二聚体内的亚基通讯。这些结果与二聚体解旋酶活性的拟议亚基切换机制有关。
