Special Centre for Molecular Medicine, Jawaharlal Nehru University, New Delhi, India.
FEBS J. 2012 Jan;279(2):234-50. doi: 10.1111/j.1742-4658.2011.08418.x. Epub 2011 Dec 9.
Replicative helicases are major motor proteins essential for chromosomal DNA replication in prokaryotes. Usually hexameric in solution, their DNA binding property must have different roles at stages ranging from the loading onto a branched structure at initiation from the origin to the highly processive translocation during elongation. Here, we have analysed the DNA binding activity of Helicobacter pylori (Hp) replicative helicase, DnaB. The results indicate that while the C-terminal region is important for its DNA binding activity, the N-terminus appears to dampen the protein's affinity for DNA. The masking activity of the N-terminus does not require ATP or hexamerization of HpDnaB and can be overcome by deleting the N-terminus. It can also be neutralized by engaging the N-terminus in an interaction with a partner like the C-terminus of DnaG primase. The inhibitory effect of the N-terminus on DNA binding activity is consistent with the 3D homology model of HpDnaB. Electron microscopy of the HpDnaB-ssDNA complex showed that HpDnaB preferentially bound at the ends of linear ssDNA and translocated along the DNA in the presence of ATP. These results provide an insight into the stimulatory and inhibitory effects of different regions of HpDnaB on DNA binding activities that may be central to the loading and translocation functions of DnaB helicases.
复制解旋酶是原核生物染色体 DNA 复制所必需的主要马达蛋白。在溶液中通常为六聚体，其 DNA 结合特性在从起始原点加载到分支结构的起始阶段到延伸过程中的高持续性易位阶段必须具有不同的作用。在这里，我们分析了幽门螺杆菌（Hp）复制解旋酶 DnaB 的 DNA 结合活性。结果表明，虽然 C 末端区域对其 DNA 结合活性很重要，但 N 末端似乎会降低该蛋白对 DNA 的亲和力。N 末端的掩蔽活性不需要 ATP 或 HpDnaB 的六聚化，并且可以通过删除 N 末端来克服。它还可以通过与 DnaG 引发酶的 C 末端等伴侣相互作用来中和 N 末端。N 末端对 DNA 结合活性的抑制作用与 HpDnaB 的 3D 同源模型一致。HpDnaB-ssDNA 复合物的电子显微镜显示，HpDnaB 在 ATP 存在下优先结合在线性 ssDNA 的末端，并沿 DNA 易位。这些结果提供了对 HpDnaB 不同区域对 DNA 结合活性的刺激和抑制作用的深入了解，这可能是 DnaB 解旋酶的加载和易位功能的核心。
