Nucleosome site exposure within oligonucleosomes

描述（由申请人提供）： 该项目将表征寡核小体内未催化的位点暴露和催化的核小体易位，这对于理解蛋白质如何访问染色质内的DNA这一未解决的问题至关重要。首先，将通过限制酶动力学方法来测量核小体内的测试核小体中的DNA的位点暴露。第二，当位点特异性DNA结合蛋白莱克萨与核小体结合时，将测量荧光共振能量转移对标记的测试核小体的诱导构象变化和结合速率。稳态FRET信号将量化构象变化，停流实验期间的FRET信号将确定结合速率。将用H1、核心组蛋白突变体和双链体设计的变化进行额外的实验，以确定它们对位点暴露、蛋白结合率和蛋白结合诱导的构象变化的影响。最后，FRET测量将确定染色质重塑蛋白ISW1是否增加位点固定开放速率，并了解在一个ATP催化循环期间诱导的构象变化。这些结果对于理解ISWl如何重塑染色质至关重要。

DESCRIPTION (provided by applicant): This project will characterize the uncatalyzed site exposure and catalyzed nucleosome translocation within oligonucleosomes that is critical for understanding the unresolved question of how proteins access DNA within chromatin. First, the site exposure of DNA in a test nucleosome within an oligonucleosome will be measured by the restriction enzymes kinetics method. Second, the induced conformational changes of and binding rates to a FRET pair labeled test nucleosome within an oligonucleosome will be measured as the site-specific DNA binding protein, LexA, binds to the nucleosome. Steady state FRET signals will quantify conformational changes and FRET signals during stopped flow experiments will determine binding rates. Additional experiment will be done with H1, core histone mutants and changes in the oligonucleosome design to determine their affects on site exposure, protein binding rates and protein binding induced conformational changes. Finally, FRET measurements will determine if the chromatin remodeling protein, ISWl, increases the site exposure-opening rate and to learn what are the conformational changes induced during one ATP catalytic cycle. These results are critical for understanding how ISWl remodels chromatin.