Naue Natalie, Curth Ute
Hannover Medical School, Institute for Biophysical Chemistry, Hannover, Germany.
Methods Mol Biol. 2012;922:133-49. doi: 10.1007/978-1-62703-032-8_8.
Bacterial single-stranded DNA-binding (SSB) proteins are essential for DNA metabolism, since they protect stretches of single-stranded DNA and are required for numerous crucial protein-protein interactions in DNA replication, recombination, and repair. At the lagging strand of the DNA replication fork of Escherichia coli, for example, SSB contacts not only DnaG primase but also the χ subunit of DNA polymerase III, thereby facilitating the switch between primase and polymerase activity. Here, we describe a powerful method that allows the study of interactions between SSB and its binding partners by sedimentation velocity experiments in an analytical ultracentrifuge. Whenever two molecules interact, a complex of a higher mass forms that can usually be distinguished from free binding partners by its different sedimentation behavior. As an example, we show how sedimentation velocity experiments of purified proteins can be employed to determine the binding parameters of the interaction of SSB and the χ subunit of DNA polymerase III from E. coli.
细菌单链DNA结合（SSB）蛋白对于DNA代谢至关重要，因为它们可保护单链DNA片段，并且在DNA复制、重组和修复过程中众多关键的蛋白质 - 蛋白质相互作用中发挥作用。例如，在大肠杆菌DNA复制叉的后随链上，SSB不仅与DnaG引发酶相互作用，还与DNA聚合酶III的χ亚基相互作用，从而促进引发酶和聚合酶活性之间的转换。在此，我们描述了一种强大的方法，该方法可通过分析超速离心机中的沉降速度实验来研究SSB与其结合伴侣之间的相互作用。每当两个分子相互作用时，就会形成质量更高的复合物，通常可通过其不同的沉降行为将其与游离的结合伴侣区分开来。例如，我们展示了如何利用纯化蛋白的沉降速度实验来确定大肠杆菌中SSB与DNA聚合酶III的χ亚基相互作用的结合参数。
