利用量子点标记的蛋白质在DNA绳索上移动的单分子荧光成像直接研究DNA-蛋白质相互作用。

DNA-Protein Interactions Studied Directly Using Single Molecule Fluorescence Imaging of Quantum Dot Tagged Proteins Moving on DNA Tightropes.

作者信息

Springall Luke, Inchingolo Alessio V, Kad Neil M

机构信息

School of Biosciences, University of Kent, Canterbury, CT2 7NH, UK.

出版信息

Methods Mol Biol. 2016;1431:141-50. doi: 10.1007/978-1-4939-3631-1_11.

DOI:10.1007/978-1-4939-3631-1_11

PMID:27283307

Abstract

Many protein interactions with DNA require specific sequences; however, how these sequences are located remains uncertain. DNA normally appears bundled in solution but, to study DNA-protein interactions, the DNA needs to be elongated. Using fluidics single DNA strands can be efficiently and rapidly elongated between beads immobilized on a microscope slide surface. Such "DNA tightropes" offer a valuable method to study protein search mechanisms. Real-time fluorescence imaging of these interactions provides quantitative descriptions of search mechanism at the single molecule level. In our lab, we use this method to study the complex process of nucleotide excision DNA repair to determine mechanisms of damage detection, lesion removal, and DNA excision.

摘要

许多蛋白质与DNA的相互作用需要特定序列；然而，这些序列是如何定位的仍不确定。DNA在溶液中通常呈束状，但为了研究DNA-蛋白质相互作用，需要将DNA拉长。利用流体技术，单链DNA可以在固定于显微镜载玻片表面的珠子之间高效快速地拉长。这种“DNA钢丝”为研究蛋白质搜索机制提供了一种有价值的方法。这些相互作用的实时荧光成像在单分子水平上提供了搜索机制的定量描述。在我们实验室，我们使用这种方法研究核苷酸切除DNA修复的复杂过程，以确定损伤检测、损伤去除和DNA切除的机制。