The data showed that by 90C120 min after G0 cells were returned to growth, Mcm4 reached levels slightly lower than those in proliferating cells. into S phase was delayed until these factors were re-synthesized. A longer S phase in these cells correlated with the activation of fewer origins of replication compared to G1 cells. The chromatin structure around inactive origins in G0 cells showed increased H3 occupancy and decreased nucleosome positioning compared to the same origins in G1 cells, inhibiting the origin binding of the Mcm4 subunit of Gossypol the MCM licensing factor. Thus, quiescent yeast cells are under-licensed during their re-entry into S phase. INTRODUCTION Quiescent cells reside in G0 phase, a cell cycle stage that is characterized by the cessation of cell growth and proliferation (1C3). Gossypol Quiescence is a conserved state that is common to all organisms and key Gossypol to the long-term survival of stem cells (4C6). Budding yeast, locus, which is 40 kb from the nearest origin of replication. The primers used for ChIP-qPCR are listed in Supplemental Table S7. BrdU IP-seq Sixty OD600 units of G1 arrested cells and 150 OD600 units of G0 Q cells were resuspended in 150 ml of YPD + pronase (60 g/ml) + CaCl2 (5 mM) + 0.2M HU (Hydroxyurea, Millipore-Sigma) or 250 ml of YPD + 0.2 M HU, respectively. Immediately before and at various times after release of G1 and G0 cells, 35 ml of cell culture were transferred into a pre-warmed 100-ml flask and incubated with 800 g/ml of BrdU (Sigma-Millipore) for overlapping periods of 20 min prior to harvesting. G1 released cells were collected at 25, 40, 55 and 70 min and G0 released cells were collected at 85, 100, 115, 130 and 160 min after BrdU pulse-labeling. Genomic DNA was isolated from each pulse-labelled sample by the smash and Gossypol grab protocol (27), and 0.2 mg/ml RNase A was added for 30 min at 37C, followed by addition of 0.1 mg/ml of Proteinase K for 30 min at 50C. DNA was purified with a Qiagen QIAquick PCR purification kit and its concentration was measured by an Invitrogen Qubit 2.0 Fluorometer. DNA was transferred to a 50 l Covaris microTUBE and sheared to 200C250 bp using a Covaris M220 Focused-ultrasonicator (peak power: 75W; duty factor: 10%; cycles per burst: 200; treatment time: 400 s). One microgram of Rabbit Polyclonal to FSHR sheared DNA was end-repaired and ligated to Ion Torrent-compatible barcode adapters (Ion Xpress??Barcode Adapters, Invitrogen) using an Ion Xpress Plus Fragment Library kit (Invitrogen). The adapter-ligated DNA was purified by Agencourt AMPure XP Reagent and DNA concentration was measured by Qubit. An equal concentration of each pulse-labeled G1 or G0 released DNA sample was pooled for immunoprecipitation (IP) and 20 ng of the mixture was set aside as input (IN) DNA (28). The pooled DNA was heated for 10 min at 95C, followed by a snap cooling on ice. Immunoprecipitation was performed with a 1:250 dilution of anti-BrdU antibody (GE Healthcare, RPN202) for 2h at 4C, followed by the chromatin immunoprecipitation procedure described above. The IP and Input DNA samples were amplified separately using an Ion Xpress Plus Fragment Library kit and sequenced with the Ion S5 System (ThermoFisher). The data represent the average of two independent experiments. Mcm4 ChIP-seq Six hundred OD600 units of G0 Q cells were isolated and released into YPD + 0.2 M HU for 90 and 105 min, and samples were collected. Ninety-six OD600 units of G1 arrested cells were also collected. Cells were fixed and processed for chromatin immunoprecipitation as described above, except the chromatin fragmentation step was performed in a 1-ml Covaris microTUBE and DNA was sheared to 200C250 bp using a Covaris M220 Focused-ultrasonicator (peak power: 75 W; duty factor: 10%; cycles per burst: 200; treatment time: 15 min for G1 samples; 25 min for G0 samples). Six microliters of anti-Myc antibodies were added to 1 mg of chromatin lysate and incubated overnight at 4C, followed by incubation with 30 l of Protein G Dynabeads (Invitrogen). Multiple immunoprecipitation reactions were carried out in.