Analysis of yeast chromatin structure and function

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DESCRIPTION (provided by applicant): Our overall objective is to determine how chromosome structure controls the repair of DMA double strand breaks (DSBs) by homologous recombination and how the repair machinery contends with this structure. Recombinational repair of a DSB requires members of the Rad52 group of proteins (Rad51p, Rad52p, Rad54p, RadSO, Mre11p, Xrs2p, Rad55p, Rad57p, Rad59p), and recent biochemical studies have shown that Rad54p has ATP-dependent chromatin remodeling activity. An intact recombinational repair system is essential for genome stability, and inactivation of mammalian Rad54 leads to shortened telomeres and development of cancers. Our general strategy is to use a powerful combination of biochemical and yeast molecular genetic approaches to dissect the dynamics of yeast chromatin structure during the repair of DSBs. The first objective is to investigate changes in chromatin structure that occur following DSB formation and during DMA strand invasion in yeast. These studies will exploit Chromosome Conformation Capture (3C) analysis to test whether phosphorylation of histone H2AX alters chromatin structure surrounding a DSB. In addition, HO endonuclease will be used to create a single DNA double strand break in a set of yeast strains that harbor a novel, homologous donor sequence embedded between positioned nucleosomes. In the second aim biochemical studies are described to dissect the mechanism by which Rad54p facilitates invasion of a Rad51 p nucleoprotein filament into a nucleosomal donor. The third objective describes in vivo and in vitro studies that focus on the roles of the InoSO and Swr1 chromatin remodeling complexes in controlling the dynamics of histone H2AX phosphorylation during checkpoint adaptation and DSB repair. The fourth aim will investigate the role of a novel casein kinase 2-dependent phosphorylation of histone H4 Serine 1 in DSB repair.