RIF1 AND NUCLEOPORINS PROMOTE THE CHROMATIN ASSOCIATION OF ORC

Abstract

Accurate duplication of the genome is critical to maintain genomic stability and failures in DNA replication can result in a host of diseases. DNA replication is a coordinated and tightly regulated process; however, much remains unknown regarding exactly how DNA replication is regulated. Key focal points in the regulation of DNA replication are controlling the activities of the Origin Recognition Complex (ORC) and the activation of the loaded helicase. In metazoans, it is still unclear how ORC is targeted to specific loci to initiate replication. I found that ORC2 associates with multiple subunits of the Nup107-160 subcomplex of the nuclear pore and determined that nucleoporins are strongly enriched at ORC2 genomic binding sites. Depleting the nucleoporin Elys reduces the chromatin association of ORC2. Depleting Elys also sensitizes cells to replication fork stalling, which could reflect a defect in establishing dormant replication origins. To further interrogate the regulation of DNA replication initiation, I also determined the factors that may regulate the activity of Rif1 through Drosophila embryogenesis. I discovered that Rif1 interacts both with ORC and the Nup107-160 subcomplex and that Rif1 binds to similar genomic regions as ORC2. The chromatin association of ORC2 is dependent on Rif1, and inversely, the chromatin association of Rif1 is dependent on ORC2. In summary, my work shows that both Rif1 and nucleoporins play roles in promoting the chromatin association of ORC, implicating both Rif1 and nucleoporins in the initiation of DNA replication in Drosophila.