Saccharomyces cerevisiae W303 Genome sequencing

Start sites of DNA replication are marked by the origin recognition complex (ORC), which initiates a coordinated series of biochemical events leading to Mcm2-7 helicase loading. However, the impact of the local chromatin environment on pre-Replication Complex (pre-RC) assembly and origin activation is poorly understood. Here, we utilized a micrococcal nuclease (MNase) digestion assay to "footprint" nucleosomes, transcription factors, and replication proteins throughout the Saccharomyces cerevisiae genome at multiple points in the cell cycle. We identified, at nucleotide resolution, an ORC-dependent footprint at 226 origins. A separate class of inefficient origins only exhibited an origin-specific footprint in G1, suggesting that origin efficiency and timing are established prior to pre-RC formation in G1. Cell cycle-dependent nucleosome remodelling during pre-RC assembly was another determinant of origin efficiency. Finally, unlike in vitro, the local chromatin environment restricts the loading of a single Mcm2-7 double hexamer either up or downstream of the ACS.