Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan.
Department of Chemistry, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan.
Nucleic Acids Res. 2023 Jul 21;51(13):6654-6667. doi: 10.1093/nar/gkad471.
Target search models of DNA-binding proteins in cells typically consider search mechanisms that include 3D diffusion and 1D sliding, which can be characterized by single-molecule tracking on DNA. However, the finding of liquid droplets of DNA and nuclear components in cells cast doubt on extrapolation from the behavior in ideal non-condensed DNA conditions to those in cells. In this study, we investigate the target search behavior of DNA-binding proteins in reconstituted DNA-condensed droplets using single-molecule fluorescence microscopy. To mimic nuclear condensates, we reconstituted DNA-condensed droplets using dextran and PEG polymers. In the DNA-condensed droplets, we measured the translational movement of four DNA-binding proteins (p53, Nhp6A, Fis and Cas9) and p53 mutants possessing different structures, sizes, and oligomeric states. Our results demonstrate the presence of fast and slow mobility modes in DNA-condensed droplets for the four DNA-binding proteins. The slow mobility mode capability is correlated strongly to the molecular size and the number of DNA-binding domains on DNA-binding proteins, but only moderately to the affinity to single DNA segments in non-condensed conditions. The slow mobility mode in DNA-condensed droplets is interpreted as a multivalent interaction mode of the DNA-binding protein to multiple DNA segments.
细胞中 DNA 结合蛋白的目标搜索模型通常考虑包括 3D 扩散和 1D 滑动的搜索机制，这些机制可以通过在 DNA 上进行单分子追踪来表征。然而，在细胞中发现 DNA 和核成分的液滴，使得从理想的非凝聚 DNA 条件下的行为推断到细胞中的行为变得值得怀疑。在这项研究中，我们使用单分子荧光显微镜研究了重组 DNA 凝聚液滴中 DNA 结合蛋白的目标搜索行为。为了模拟核凝聚物，我们使用葡聚糖和 PEG 聚合物重组了 DNA 凝聚液滴。在 DNA 凝聚液滴中，我们测量了四个 DNA 结合蛋白（p53、Nhp6A、Fis 和 Cas9）和具有不同结构、大小和寡聚状态的 p53 突变体的平移运动。我们的结果表明，四种 DNA 结合蛋白在 DNA 凝聚液滴中存在快速和慢速迁移模式。慢速迁移模式的能力与 DNA 结合蛋白的分子大小和 DNA 结合域的数量强烈相关，但与非凝聚条件下单个 DNA 片段的亲和力中度相关。DNA 凝聚液滴中的慢速迁移模式被解释为 DNA 结合蛋白与多个 DNA 片段的多价相互作用模式。
