Project Summary/Abstract In eukaryotes, chromatin replication is responsible for the transmission of genetic and epigenetic information between daughter cells. Interfering with this process can lead to genetic and/or epigenetic mutations, which are both causative for many human diseases, including cancer and neurodegenerative disorders. Although epigenetic features like histone variants, histone modifications and DNA methylation need to be properly copied during chromatin replication, they also serve important roles in regulating DNA replication itself, as well as other cellular processes occurring simultaneously like transcription and DNA repair. Our research group seeks to understand the contribution of epigenetic information in regulating these processes during chromatin replication. We are particularly interested in the roles played by conserved histone H3 variants in maintaining genomic and epigenomic stability. Recently, we demonstrated that the replication-dependent histone H3.1 variant, which is conserved in all multicellular organisms, directly regulates TSK/TONSL-mediated DNA repair to resolve stalled and broken replication forks. This discovery revealed the first protein domain (the tetratricopeptide domain of TSK/TONSL) that can “read” H3.1 to mediate a specific function at chromatin (i.e., DNA repair). One of our goals over the next five years is to understand the impact of the H3.1-TSK/TONSL pathway in protecting the genome against replication-associated mutations. We are also interested in elucidating the molecular mechanisms by which TSK/TONSL initiates homologous recombination-mediated DNA repair during replication. Another related goal is to uncover and functionally characterize other proteins that are regulated by the conserved H3.1 variant. Finally, we aim to explore if the H3.1-TSK/TONSL interaction plays additional roles during chromatin replication aside from DNA repair. The proposed research will reveal novel functions for histone H3 variants. In the long term, our work will provide a better understanding of the multiple roles played by chromatin-based molecular mechanisms in promoting the maintenance of genetic and epigenetic information. Protecting this information during replication is critical for proper development in multicellular eukaryotes, and for preventing the occurrence and accumulation of mutations that can negatively affect human health.