Fluorescence approaches for biochemical analysis of ATP-dependent chromatin remodeling enzymes.
Fluorescence approaches for biochemical analysis of ATP-dependent chromatin remodeling enzymes.
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DOI：
10.1016/bs.mie.2022.02.024
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发表时间：
2022
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染色质的动态性质是调控基因表达的重要机制。染色质由核小体组成，核小体是由DNA包裹的组蛋白八聚体，这些结构的操纵是由称为ATP依赖性染色质重塑酶的蛋白质家族进行的。这些酶进行各种各样的活动，从适当地定位和调节核小体在基因上的密度，到安装和去除序列变体的组蛋白，再到从DNA中排出。这些活性在染色质结构的适当维持中具有关键作用，并且染色质重塑的失调与各种疾病的病理生理学直接相关。因此，对染色质重塑酶的机制理解是期望的，既作为这种基本细胞活性的驱动者，又作为疾病中潜在的新型治疗靶点。在这一章中，我们涵盖了我们目前的重塑底物结合亲和力和催化活性的表征方法，利用荧光偏振和Förster共振能量转移测定。
The dynamic nature of chromatin is an essential mechanism by which gene expression is regulated. Chromatin is comprised of nucleosomes, an octamer of histone proteins wrapped by DNA, and manipulation of these structures is carried out by a family of proteins known as ATP-dependent chromatin remodeling enzymes. These enzymes carry out a diverse range of activities, from appropriately positioning and adjusting the density of nucleosomes on genes, to installation and removal of histones for sequence variants, to ejection from DNA. These activities have a critical role in the proper maintenance of chromatin architecture, and dysregulation of chromatin remodeling is directly linked to the pathophysiology of various diseases. Mechanistic understanding of chromatin remodeling enzymes is therefore desirable, both as the drivers of this essential cellular activity and as potentially novel therapeutic targets in disease. In this chapter we cover our current methods for characterization of remodeler substrate binding affinity and catalytic activity, leveraging fluorescence polarization and Förster resonance energy transfer assay.