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 by the non-homologous end joining (NHEJ) pathway in mammalian cells. If repaired incorrectly, DSBs result in abberations 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 a novel chromatin assembly factor, termed ASF1, essential for the repair of radiation-induced, endogenous and developmentally-programmed DNA damage in vivo. Surprisingly, ASF1 is even required for DNA repair processes, such as NHEJ, that do not invoke the assembly of chromatin onto newly-synthesized DNA. We will test the hypothesis that chromatin structure is altered by the ASF1 chromatin assembly factor during NHEJ and that changes to the chromatin structure are essential and intrinsic to the repair of DNA damage. In order to gain insight into the repair of radiation-induced DNA damage, the approach will be to induce a highly specific endonuclease within yeast to generate a synchronous defined DSB that can only be repaired by NHEJ. Using this model system, we will map for the first time the changes to the chromatin structure that precede, accompany and follow the repair of a DSB by NHEJ. Finally, we will determine the generality of the influence of chromatin structure on NHEJ, by examining the role of ASF1 and chromatin structure in mammalian cells. The findings of the proposed experiments using model genetic systems to induce a defined DSB will provide the foundation for understanding the fundamental, yet previously overlooked, 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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