The goal of this research is to define the fundamental role that chromatin plays during the repair of radiation-induced double-strand breaks (DSBs). Cell survival and maintenance of genome integrity are critically dependent on the repair of DSBs. If repaired incorrectly, DSBs result in aberrations such as chromosomal rearrangements and can lead to formation of cancers. In order to fully understand the repair of radiation-induced DSBs, it is important to consider the natural context - chromatin. The packaging of the genome into chromatin is likely to influence DNA repair processes by analogy to the situation with gene expression. Accordingly, we have discovered that the chromatin assembly factors Asf1 and CAF-1 are essential for cell survival following the repair of radiation-induced, endogenous and developmentally-programmed DNA damage in vivo. Furthermore, we have recently shown for the first time that histone acetylation changes locally during DSB repair via the homologous recombination pathway, and that cells die if they cannot change their acetylation state during DSB repair. We will test the hypothesis that specific post- translational modifications of histones, remodeling, disassembly, and reassembly of the chromatin occur at the DSB during repair. Furthermore, we will define the molecular mechanism as to why these chromatin dynamics are essential and intrinsic to chromosomal repair. Finally, we will identify novel chromatin modifications that are critical for DSB repair. In order to gain insight into the repair of radiation-induced DNA damage, the approach will be to synchronously induce a highly specific endonuclease within yeast to study the molecular events at a unique defined DSB. Using this model system, we will map the changes to the chromatin structure that precede, accompany and follow the repair of a DSB. The findings of the proposed experiments will provide the foundation for understanding the fundamental, yet poorly understood, role of chromatin structure during the repair of radiation-induced DSBs. As such, these studies are directly applicable to human diseases that result from radiation-induced loss of genome integrity, including many forms of cancer.
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