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

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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.