Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO 63110
Proc Natl Acad Sci U S A. 2019 Aug 13;116(33):16320-16325. doi: 10.1073/pnas.1905513116. Epub 2019 Jul 30.
UvrD is a superfamily 1 helicase/translocase that functions in DNA repair, replication, and recombination. Although a UvrD monomer can translocate along single-stranded DNA, self-assembly or interaction with an accessory protein is needed to activate its helicase activity in vitro. Our previous studies have shown that an MutL dimer can activate the UvrD monomer helicase in vitro, but the mechanism is not known. The UvrD 2B subdomain is regulatory and can exist in extreme rotational conformational states. By using single-molecule FRET approaches, we show that the 2B subdomain of a UvrD monomer bound to DNA exists in equilibrium between open and closed states, but predominantly in an open conformation. However, upon MutL binding to a UvrD monomer-DNA complex, a rotational conformational state is favored that is intermediate between the open and closed states. Parallel kinetic studies of MutL activation of the UvrD helicase and of MutL-dependent changes in the UvrD 2B subdomain show that the transition from an open to an intermediate 2B subdomain state is on the pathway to helicase activation. We further show that MutL is unable to activate the helicase activity of a chimeric UvrD containing the 2B subdomain of the structurally similar Rep helicase. Hence, MutL activation of the monomeric UvrD helicase is regulated specifically by its 2B subdomain.
UvrD 是一个超家族 1 螺旋酶/转位酶，在 DNA 修复、复制和重组中发挥作用。虽然 UvrD 单体可以沿着单链 DNA 迁移，但需要自我组装或与辅助蛋白相互作用才能在体外激活其螺旋酶活性。我们之前的研究表明，MutL 二聚体可以在体外激活 UvrD 单体的螺旋酶活性，但机制尚不清楚。UvrD 的 2B 亚基域具有调节作用，可以存在于极端旋转构象状态。通过使用单分子 FRET 方法，我们表明与 DNA 结合的 UvrD 单体的 2B 亚基域在打开和关闭状态之间处于平衡，但主要处于打开构象。然而，当 MutL 与 UvrD 单体-DNA 复合物结合时，有利于一种介于打开和关闭状态之间的旋转构象状态。MutL 对 UvrD 螺旋酶的激活和 MutL 依赖性 UvrD 2B 亚基域变化的平行动力学研究表明，从开放状态到中间 2B 亚基域状态的转变是螺旋酶激活的途径。我们进一步表明，MutL 无法激活包含结构相似的 Rep 螺旋酶 2B 亚基域的嵌合 UvrD 的螺旋酶活性。因此，MutL 对单体 UvrD 螺旋酶的激活受到其 2B 亚基域的特异性调节。
