The Role of Histone Chaperones in Genomic Stability – ASFGENOMSTAB

The organization of DNA around nucleosomes profoundly affects its accessibility to enzymes involved in gene expression, DNA replication, DNA repair, and DNA recombination, and defects in chromatin organization or modification can cause disease by affecting genome expression and stability. Nucleosomes contain histones, a set of highly basic proteins whose association and dissociation from DNA is mediated by histone chaperones. The number of identified histone chaperones has been increasing, but their crucial importance in all aspects of DNA metabolism has only recently begun to be recognized. Our long-term goal is to identify a complete set of histone chaperones in the model organism Saccharomyces cerevisiae, and to elucidate their roles in genome stability and the cellular response to DNA damage. - Our long-standing interest in cell cycle regulation and checkpoints led us to study Asf1 (anti-silencing factor 1), a highly conserved chaperone of the histone H3/H4 complex. We identified Asf1 in a genetic screen for proteins interacting with the Rad53 checkpoint kinase in budding yeast, and we carried out an extensive structure-function analysis of the yeast and human Asf1 proteins. Our central hypothesis is that histone chaperones such as Asf1 contribute to DNA metabolism in at least three different ways. First, they participate in assembling and disassembling histones in order to make chromatin more accessible to DNA metabolic enzymes and to restore the structure of the chromatin after the action of these enzymes. Second, they contribute to the recruitment of other regulatory proteins to chromatin. Third, they act as cofactors in the post-translational modification of histones. We propose two specific aims to test this hypothesis: - 1. To isolate mutant yeast strains in which the interaction of Asf1 with specific protein partners is disrupted and to characterize their functional defects. A large number of interacting partners have been identified for Asf1, but it is not known how these contribute to its functions in genome stability and the cellular response to DNA damage. We determined the solution structure of the functional N-terminal domain of Asf1 by NMR in collaboration with Françoise Ochsenbein, an NMR structural biologist in our department. A combination of genetic, biochemical, and NMR techniques were used to identify human and yeast Asf1 mutants with histone-binding defects. Thus work revealed the crucial role of the Asf1-H3 interaction for all of Asf1's major functions in yeast. The Rad53 checkpoint kinase is another notable partner of Asf1 in conditions of normal cell growth. However, this complex dissociates when cells are subjected to genotoxic stress. This regulated interaction strongly suggests that it plays a role in the cellular response to genotoxic stress. We will identify mutants that specifically disrupt the interaction of Asf1 with the Rad53 kinase using approaches similar to those that allowed us to successfully disrupt the Asf1-H3/H4 interaction. These mutants will then be characterized phenotypically in order to determine the functional role of the Asf1-Rad53 interaction. - 2. To identify novel DNA replication-coupled nucleosome assembly pathways that function in the absence of CAF-I and Asf1. Nucleosomes must be efficiently reassembled on newly synthesized DNA to ensure genomic stability. CAF-I and Asf1 are two major histone chaperones that have been implicated in DNA-replication coupled nucleosome assembly in somatic cells. Surprisingly, simultaneous inactivation of the CAF-I and Asf1 histone chaperones is not lethal in S. cerevisiae. We will use both biochemical and genetic approaches to identify the histone H3/H4 chaperones that function in replication-coupled nucleosome assembly in the absence of CAF-I and Asf1. - - These systematic studies will drive the field forward by identifying the full set of histone chaperones required for replication-coupled nucleosome assembly and by providing a molecular u...