Lovullo D, Daniel D, Yodh J, Lohr D, Woodbury N W
Department of Chemistry and Biochemistry, Arizona State University, Tempe, AZ 85287, USA.
Anal Biochem. 2005 Jun 1;341(1):165-72. doi: 10.1016/j.ab.2005.03.022.
Nucleosomes are the basic units of eukaryotic chromatin structure. By restricting factor access to regulatory DNA sequences, nucleosomes significantly impact genomic processes such as transcription, and various mechanisms to alter nucleosome structure to relieve this repression have evolved. Both nucleosomes and processes that alter them are inherently dynamic in nature. Thus, studies of dynamics will be necessary to truly understand these relief mechanisms. We describe here the characteristics of a novel fluorescence resonance energy transfer-based reporter that can clearly signal the formation of a canonical nucleosome structure and follow conformational and compositional changes in that structure, both at the ensemble-average (bulk) and at the single molecule level. Labeled nucleosomes behave conformationally and thermodynamically like typical nucleosomes; thus they are relevant reporters of nucleosome behavior. Nucleosomes and free DNA are readily distinguishable at the single-molecule level. Thus, these labeled nucleosomes are well suited to studies of dynamic changes in nucleosome structure including single-molecule dynamics.
核小体是真核染色质结构的基本单位。通过限制因子对调控DNA序列的 access，核小体显著影响转录等基因组过程，并且已经进化出各种改变核小体结构以减轻这种抑制的机制。核小体以及改变它们的过程本质上都是动态的。因此，对动力学的研究对于真正理解这些缓解机制是必要的。我们在此描述一种基于新型荧光共振能量转移的报告分子的特性，它可以清晰地指示经典核小体结构的形成，并在整体平均（总体）和单分子水平上跟踪该结构的构象和组成变化。标记的核小体在构象和热力学上表现得像典型的核小体；因此它们是核小体行为的相关报告分子。核小体和游离DNA在单分子水平上很容易区分。因此，这些标记的核小体非常适合用于研究核小体结构的动态变化，包括单分子动力学。
