Genome-wide chromatin footprinting reveals changes in replication origin architecture induced by pre-RC assembly

• Published in: Genes & development Vol. 29; no. 2; pp. 212 - 224
• Main Authors: Belsky, Jason A, MacAlpine, Heather K, Lubelsky, Yoav, Hartemink, Alexander J, MacAlpine, David M
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 15.01.2015
• Subjects: Cell Cycle - genetics; Chromatin - genetics; G1 Phase - genetics; G2 Phase - genetics; Genome-Wide Association Study; Minichromosome Maintenance Proteins - metabolism; Nucleosomes - metabolism; Origin Recognition Complex - metabolism; Research Paper; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; nucleosome; DNA replication; chromatin; origin recognition complex (ORC)
• ISSN: 0890-9369; 1549-5477
• DOI: 10.1101/gad.247924.114

Start sites of DNA replication are marked by the origin recognition complex (ORC), which coordinates Mcm2-7 helicase loading to form the prereplicative complex (pre-RC). Although pre-RC assembly is well characterized in vitro, the process is poorly understood within the local chromatin environment surrounding replication origins. To reveal how the chromatin architecture modulates origin selection and activation, we "footprinted" nucleosomes, transcription factors, and replication proteins at multiple points during the Saccharomyces cerevisiae cell cycle. Our nucleotide-resolution protein occupancy profiles resolved a precise ORC-dependent footprint at 269 origins in G2. A separate class of inefficient origins exhibited protein occupancy only in G1, suggesting that stable ORC chromatin association in G2 is a determinant of origin efficiency. G1 nucleosome remodeling concomitant with pre-RC assembly expanded the origin nucleosome-free region and enhanced activation efficiency. Finally, the local chromatin environment restricts the loading of the Mcm2-7 double hexamer either upstream of or downstream from the ARS consensus sequence (ACS).