Macromolecular Biochemistry, Leiden Institute of Chemistry, Leiden University, Leiden 2333CC, The Netherlands.
Centre for Microbial Cell Biology, Leiden University, Leiden, The Netherlands.
Nucleic Acids Res. 2022 Jan 25;50(2):e10. doi: 10.1093/nar/gkab993.
The interplay between three-dimensional chromosome organisation and genomic processes such as replication and transcription necessitates in vivo studies of chromosome dynamics. Fluorescent organic dyes are often used for chromosome labelling in vivo. The mode of binding of these dyes to DNA cause its distortion, elongation, and partial unwinding. The structural changes induce DNA damage and interfere with the binding dynamics of chromatin-associated proteins, consequently perturbing gene expression, genome replication, and cell cycle progression. We have developed a minimally-perturbing, genetically encoded fluorescent DNA label consisting of a (photo-switchable) fluorescent protein fused to the DNA-binding domain of H-NS - a bacterial nucleoid-associated protein. We show that this DNA label, abbreviated as HI-NESS (H-NS-based indicator for nucleic acid stainings), is minimally-perturbing to genomic processes and labels chromosomes in eukaryotic cells in culture, and in zebrafish embryos with preferential binding to AT-rich chromatin.
三维染色体组织与复制和转录等基因组过程之间的相互作用需要对染色体动力学进行体内研究。荧光有机染料常用于体内染色体标记。这些染料与 DNA 的结合方式导致 DNA 的扭曲、伸长和部分解旋。结构变化会导致 DNA 损伤，并干扰染色质相关蛋白的结合动力学，从而扰乱基因表达、基因组复制和细胞周期进程。我们开发了一种最小干扰的、基因编码的荧光 DNA 标记，由（光可切换的）荧光蛋白与 H-NS 的 DNA 结合域融合而成 - 一种细菌核相关蛋白。我们表明，这种 DNA 标记，缩写为 HI-NESS（基于 H-NS 的核酸染色指示剂），对基因组过程的干扰最小，并在培养的真核细胞和斑马鱼胚胎中标记染色体，优先与富含 AT 的染色质结合。
