Department of Biological Sciences, Columbia University, New York, NY 10032, USA.
Proc Natl Acad Sci U S A. 2012 Nov 6;109(45):E3074-83. doi: 10.1073/pnas.1211364109. Epub 2012 Sep 24.
The ability of proteins to locate specific targets among a vast excess of nonspecific DNA is a fundamental theme in biology. Basic principles governing these search mechanisms remain poorly understood, and no study has provided direct visualization of single proteins searching for and engaging target sites. Here we use the postreplicative mismatch repair proteins MutSα and MutLα as model systems for understanding diffusion-based target searches. Using single-molecule microscopy, we directly visualize MutSα as it searches for DNA lesions, MutLα as it searches for lesion-bound MutSα, and the MutSα/MutLα complex as it scans the flanking DNA. We also show that MutLα undergoes intersite transfer between juxtaposed DNA segments while searching for lesion-bound MutSα, but this activity is suppressed upon association with MutSα, ensuring that MutS/MutL remains associated with the damage-bearing strand while scanning the flanking DNA. Our findings highlight a hierarchy of lesion- and ATP-dependent transitions involving both MutSα and MutLα, and help establish how different modes of diffusion can be used during recognition and repair of damaged DNA.
蛋白质在大量非特异性 DNA 中定位特定靶标的能力是生物学中的一个基本主题。控制这些搜索机制的基本原则仍未得到很好的理解，并且没有研究提供过对单个蛋白质搜索和结合靶标位点的直接可视化。在这里，我们使用复制后错配修复蛋白 MutSα 和 MutLα 作为理解基于扩散的靶标搜索的模型系统。使用单分子显微镜，我们直接可视化 MutSα 搜索 DNA 损伤，MutLα 搜索与损伤结合的 MutSα，以及 MutSα/MutLα 复合物扫描侧翼 DNA。我们还表明，MutLα 在搜索与损伤结合的 MutSα 时会在相邻 DNA 片段之间进行位点间转移，但这种活性在与 MutSα 结合时受到抑制，从而确保 MutS/MutL 在扫描侧翼 DNA 时仍然与带有损伤的链结合。我们的发现强调了涉及 MutSα 和 MutLα 的一系列与损伤和 ATP 依赖性的转变，有助于确定在识别和修复受损 DNA 时可以使用哪些不同的扩散模式。
