The role of CENP-A and centromere components in DNA repair

描述（由申请人提供）：染色体是遗传的基本单位，在细胞分裂过程中染色体正确分离到子细胞对于防止可能导致癌症或不孕症等疾病的错误至关重要。着丝粒是染色体上的一个区域，它介导这种分离，并通过稳定地获得主要的表观遗传标记CENP-A（一种专门在着丝粒上发现的组蛋白H3变体）来决定表观遗传。克利夫兰实验室先前已经证明，CENP-A可以迅速重新定位到DNA损伤的位置，并暂时停留在那里。现在，我将确定CENP-A在DNA损伤部位的瞬时招募的特征，并确定它是否有助于整体DNA修复。通过使用工程核酸酶创建位点特异性双链断裂，我将使用荧光标记蛋白质或免疫荧光的活细胞成像和已知定位于DNA损伤的蛋白质（包括53BP1）共染色来确定CENP-A（及其伴侣蛋白）的加载速度。我将使用细胞同步技术来确定DNA损伤位点对CENP-A定位的细胞周期依赖性以及这种CENP-A粒子（八聚体、六聚体、半聚体）的性质。此外，我将使用染色质免疫沉淀，定量实时PCR和/或DNA测序，通过CENP-A和CENP-A相关蛋白确定染色质重塑的程度。最后，通过免疫荧光和染色质免疫沉淀，我将确定CENP-A相关蛋白和DNA损伤蛋白的募集是否依赖于CENP-A。

DESCRIPTION (provided by applicant): Chromosomes are the basic unit of inheritance and the proper segregation of chromosomes to daughter cells during cell division is essential to prevent errors that can lead to diseases such as cancer or infertility. The centromere is a region of the chromosome that mediates this segregation and is determined epigenetically through the stable acquisition of the prime epigenetic mark, CENP-A, a histone H3 variant specifically found at centromeres. The Cleveland lab has previously demonstrated that CENP-A is rapidly re-localized to sites of DNA damage and to remain there transiently. I will now determine the characteristics of transient recruitment of CENP-A to DNA damage sites and determines if it facilitates overall DNA repair. By creating a site specific double strand break with engineered nucleases, I will determine how quickly CENP-A (and its partner proteins) is loaded using live cell imaging of fluorescently tagged proteins or immunofluorescence and co-staining with proteins already known to be localized to DNA damage (including 53BP1). I will use cell synchronization techniques to determine the cell cycle dependency of CENP-A localization to sites of DNA damage and the nature of this CENP-A particle (octamer, hexamer, hemisome). In addition, I will determine the extent of chromatin remodeling by CENP-A and CENP-A associated proteins using chromatin immunoprecipitation followed by quantitative real time PCR and/or DNA sequencing. Finally, by using immunofluorescence and chromatin immunoprecipiation I will determine if the recruitment of CENP-A associated proteins and DNA damage proteins is dependent on CENP-A.