Control of origin licensing in euchromatin by the yeast heterochromatin regulators Sir2 and Sir3

Prior to the initiation of DNA synthesis, ORC and Cdc6 load the MCM helicase at all replication origins. However, when MCM loading is compromised (e.g., as in the temperature-sensitive cdc6-4 mutant) the heterochromatin regulator Sir2 effectively blocks MCM loading at many euchromatic origins by an unknown mechanism. Here we show that the Sir2 and Sir3 heterochromatin proteins act as direct negative regulators of MCM loading at the majority of euchromatic origins. Nucleosomes surrounding the Sir2-regulated origins bound both Sir2 and Sir3 and were specifically depleted for H4K16 acetylation in a SIR2-dependent manner in wild type cells. Furthermore, histone H3 and H4 mutants that disrupt Sir3-nucleosome binding also rescued the cdc6-4 growth temperature-sensitive growth defect. These findings expand the role of heterochromatin regulators in DNA replication and strongly suggest that many euchromatic origins act as nucleators of Sir2-3 chromatin that in turn attenuates MCM loading.