Biophysics Program, University of California, Berkeley, Berkeley, CA 94720-3220, USA.
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.
Mol Cell. 2013 Dec 26;52(6):844-54. doi: 10.1016/j.molcel.2013.11.016.
Cellular replication forks are powered by ring-shaped, hexameric helicases that encircle and unwind DNA. To better understand the molecular mechanisms and control of these enzymes, we used multiple methods to investigate the bacterial replicative helicase, DnaB. A 3.3 Å crystal structure of Aquifex aeolicus DnaB, complexed with nucleotide, reveals a newly discovered conformational state for this motor protein. Electron microscopy and small angle X-ray scattering studies confirm the state seen crystallographically, showing that the DnaB ATPase domains and an associated N-terminal collar transition between two physical states in a nucleotide-dependent manner. Mutant helicases locked in either collar state are active but display different capacities to support critical activities such as duplex translocation and primase-dependent RNA synthesis. Our findings establish the DnaB collar as an autoregulatory hub that controls the ability of the helicase to transition between different functional states in response to both nucleotide and replication initiation/elongation factors.
细胞复制叉由环形六聚体解旋酶驱动，这些解旋酶环绕并解开 DNA。为了更好地理解这些酶的分子机制和调控，我们使用多种方法研究了细菌复制解旋酶 DnaB。来自水生栖热菌（Aquifex aeolicus）的 DnaB 与核苷酸形成的复合物的 3.3Å 晶体结构揭示了这种马达蛋白的一个新发现的构象状态。电子显微镜和小角度 X 射线散射研究证实了晶体学中观察到的状态，表明 DnaB ATP 酶结构域和相关的 N 端环在核苷酸依赖性的方式下在两种物理状态之间转换。锁定在环状态的突变解旋酶仍然具有活性，但显示出不同的能力来支持关键活性，如双链体易位和引物酶依赖性 RNA 合成。我们的发现确立了 DnaB 环作为一个自动调节中心，控制解旋酶在核苷酸和复制起始/延伸因子的作用下，在不同功能状态之间转换的能力。
